Conjugates and compositions for cellular delivery

ABSTRACT

This invention features conjugates, degradable linkers, compositions, methods of synthesis, and applications thereof, including cholesterol, folate, galactose, galactosamine, N-acetyl galactosamine, PEG, phospholipid, peptide and human serum albumin (HSA) derived conjugates of biologically active compounds, including antibodies, antivirals, chemotherapeutics, peptides, proteins, hormones, nucleosides, nucleotides, non-nucleosides, and nucleic acids including enzymatic nucleic acids, DNAzymes, allozymes, antisense, dsRNA, siNA, siRNA, triplex oligonucleotides, 2,5-A chimeras, decoys and aptamers.

This patent application is a continuation of U.S. patent applicationSer. No. 10/780,447, filed on Feb. 13, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/427,160,filed Apr. 30, 2003, which is a continuation-in-part of InternationalPatent Application No. PCT/US02/15876, filed May 17, 2002, that claimsthe benefit of U.S. Provisional Application No. 60/292,217, filed May18, 2001, U.S. Provisional Application No. 60/362,016, filed Mar. 6,2002, U.S. Provisional Application No. 60/306,883, filed Jul. 20, 2001,and U.S. Provisional Application No. 60/311,865, filed Aug. 13, 2001;and parent U.S. patent application Ser. No. 10/780,447 is also acontinuation-in-part of International Patent Application No.PCT/US03/05346, filed Feb. 20, 2003, and International PatentApplication No. PCT/US03/05028, filed Feb. 20, 2003, both of which claimthe benefit of U.S. Provisional Application No. 60/358,580, filed Feb.20, 2002, U.S. Provisional Application No. 60/363,124, filed Mar. 11,2002, of U.S. Provisional Application No. 60/386,782, filed Jun. 6,2002, U.S. Provisional Application No. 60/406,784, filed Aug. 29, 2002,U.S. Provisional Application No. 60/408,378, filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293, filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129, filed Jan. 15, 2003. Theseapplications are hereby incorporated by reference herein in theirentirety including the drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file“SequenceListingUpdated12May 20093USCNT2,” created on May 12, 2009,which is 10,658 bytes in size.

BACKGROUND OF THE INVENTION

The present invention relates to conjugates, compositions, methods ofsynthesis, and applications thereof. The discussion is provided only forunderstanding of the invention that follows. This summary is not anadmission that any of the work described below is prior art to theclaimed invention.

The cellular delivery of various therapeutic compounds, such asantiviral and chemotherapeutic agents, is usually compromised by twolimitations. First the selectivity of therapeutic agents is often low,resulting in high toxicity to normal tissues. Secondly, the traffickingof many compounds into living cells is highly restricted by the complexmembrane systems of the cell. Specific transporters allow the selectiveentry of nutrients or regulatory molecules, while excluding mostexogenous molecules such as nucleic acids and proteins. Variousstrategies can be used to improve transport of compounds into cells,including the use of lipid carriers and various conjugate systems.Conjugates are often selected based on the ability of certain moleculesto be selectively transported into specific cells, for example viareceptor mediated endocytosis. By attaching a compound of interest tomolecules that are actively transported across the cellular membranes,the effective transfer of that compound into cells or specific cellularorganelles can be realized. Alternately, molecules that are able topenetrate cellular membranes without active transport mechanisms, forexample, various lipophilic molecules, can be used to deliver compoundsof interest. Examples of molecules that can be utilized as conjugatesinclude but are not limited to peptides, hormones, fatty acids,vitamins, flavonoids, sugars, reporter molecules, reporter enzymes,chelators, porphyrins, intercalcators, and other molecules that arecapable of penetrating cellular membranes, either by active transport orpassive transport.

The delivery of compounds to specific cell types, for example, cancercells or cells specific to particular tissues and organs, can beaccomplished by utilizing receptors associated with specific cell types.Particular receptors are overexpressed in certain cancerous cells,including the high affinity folic acid receptor. For example, the highaffinity folate receptor is a tumor marker that is overexpressed in avariety of neoplastic tissues, including breast, ovarian, cervical,colorectal, renal, and nasoparyngeal tumors, but is expressed to a verylimited extent in normal tissues. The use of folic acid based conjugatesto transport exogenous compounds across cell membranes can provide atargeted delivery approach to the treatment and diagnosis of disease andcan provide a reduction in the required dose of therapeutic compounds.Furthermore, therapeutic bioavialability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates, including folate bioconjugates. Godwin et al., 1972, J.Biol. Chem., 247, 2266-2271, report the synthesis of biologically activepteroyloligo-L-glutamates. Habus et al., 1998, Bioconjugate Chem., 9,283-291, describe a method for the solid phase synthesis of certainoligonucleotide-folate conjugates. Cook, U.S. Pat. No. 6,721,208,describes certain oligonucleotides modified with specific conjugategroups. The use of biotin and folate conjugates to enhance transmembranetransport of exogenous molecules, including specific oligonucleotideshas been reported by Low et al., U.S. Pat. Nos. 5,416,016, 5,108,921,and International PCT publication No. WO 90/12096. Manoharan et al.,International PCT publication No. WO 99/66063 describe certain folateconjugates, including specific nucleic acid folate conjugates with aphosphoramidite moiety attached to the nucleic acid component of theconjugate, and methods for the synthesis of these folate conjugates.Nomura et al., 2000, J. Org. Chem., 65, 5016-5021, describe thesynthesis of an intermediate,alpha-[2-(trimethylsilyl)ethoxycarbonl]folic acid, useful in thesynthesis of certain types of folate-nucleoside conjugates. Guzaev etal., U.S. Pat. No. 6,335,434, describes the synthesis of certain folateoligonucleotide conjugates.

The delivery of compounds to other cell types can be accomplished byutilizing receptors associated with a certain type of cell, such ashepatocytes. For example, drug delivery systems utilizingreceptor-mediated endocytosis have been employed to achieve drugtargeting as well as drug-uptake enhancement. The asialoglycoproteinreceptor (ASGPr) (see for example Wu and Wu, 1987, J. Biol. Chem. 262,4429-4432) is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins, such as asialoorosomucoid (ASOR).Binding of such glycoproteins or synthetic glycoconjugates to thereceptor takes place with an affinity that strongly depends on thedegree of branching of the oligosaccharide chain, for example,triatennary structures are bound with greater affinity than biatenarryor monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee and Lee, 1987,Glycoconjugate J., 4, 317-328, obtained this high specificity throughthe use of N-acetyl-D-galactosamine as the carbohydrate moiety, whichhas higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose andgalactosamine based conjugates to transport exogenous compounds acrosscell membranes can provide a targeted delivery approach to the treatmentof liver disease such as HBV and HCV infection or hepatocellularcarcinoma. The use of bioconjugates can also provide a reduction in therequired dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavialability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use ofbioconjugates.

A number of peptide based cellular transporters have been developed byseveral research groups. These peptides are capable of crossing cellularmembranes in vitro and in vivo with high efficiency. Examples of suchfusogenic peptides include a 16-amino acid fragment of the homeodomainof ANTENNAPEDIA, a Drosophila transcription factor (Wang et al., 1995,PNAS USA., 92, 3318-3322); a 17-mer fragment representing thehydrophobic region of the signal sequence of Kaposi fibroblast growthfactor with or without NLS domain (Antopolsky et al., 1999, Bioconj.Chem., 10, 598-606); a 17-mer signal peptide sequence of caimancrocodylus Ig(5) light chain (Chaloin et al., 1997, Biochem. Biophys.Res. Comm., 243, 601-608); a 17-amino acid fusion sequence of HIVenvelope glycoprotein gp4114, (Morris et al., 1997, Nucleic Acids Res.,25, 2730-2736); the HIV-1 Tat49-57 fragment (Schwarze et al., 1999,Science, 285, 1569-1572); a transportan A—achimeric 27-mer consisting ofN-terminal fragment of neuropeptide galanine and membrane interactingwasp venom peptide mastoporan (Lindgren et al., 2000, BioconjugateChem., 11, 619-626); and a 24-mer derived from influenza virushemagglutinin envelop glycoprotein (Bongartz et al., 1994, Nucleic AcidsRes., 22, 4681-4688).

These peptides were successfully used as part of an antisenseoligonucleotide-peptide conjugate for cell culture transfection withoutlipids. In a number of cases, such conjugates demonstrated better cellculture efficacy then parent oligonucleotides transfected using lipiddelivery. In addition, use of phage display techniques has identifiedseveral organ targeting and tumor targeting peptides in vivo (Ruoslahti,1996, Ann. Rev. Cell Dev. Biol., 12, 697-715). Conjugation of tumortargeting peptides to doxorubicin has been shown to significantlyimprove the toxicity profile and has demonstrated enhanced efficacy ofdoxorubicin in the in vivo murine cancer model MDA-MB-435 breastcarcinoma (Arap et al., 1998, Science, 279, 377-380).

Hudson et al., 1999, Int. J. Pharm., 182, 49-58, describes the cellulardelivery of specific hammerhead ribozymes conjugated to a transferrinreceptor antibody. Janjic et al., U.S. Pat. No. 6,168,778, describesspecific VEGF nucleic acid ligand complexes for targeted drug delivery.Bonora et al., 1999, Nucleosides Nucleotides, 18, 1723-1725, describesthe biological properties of specific antisense oligonucleotidesconjugated to certain polyethylene glycols. Davis and Bishop,International PCT publication No. WO 99/17120 and Jaeschke et al., 1993,Tetrahedron Lett., 34, 301-4 describe specific methods of preparingpolyethylene glycol conjugates. Tullis, International PCT PublicationNo. WO 88/09810; Jaschke, 1997, ACS Sympl Ser., 680, 265-283; Jaschke etal., 1994, Nucleic Acids Res., 22, 4810-17; Efimov et al., 1993, Bioorg.Khim., 19, 800-4; and Bonora et al., 1997, Bioconjugate Chem., 8,793-797, describe specific oligonucleotide polyethylene glycolconjugates. Manoharan, International PCT Publication No. WO 00/76554,describes the preparation of specific ligand-conjugatedoligodeoxyribonucleotides with certain cellular, serum, or vascularproteins. Defrancq and Lhomme, 2001, Bioorg Med Chem. Lett., 11,931-933; Cebon et al., 2000, Aust. J. Chem., 53, 333-339; and Salo etal., 1999, Bioconjugate Chem., 10, 815-823 describe specific aminooxypeptide oligonucleotide conjugates.

SUMMARY OF THE INVENTION

The present invention features compositions and conjugates to facilitatedelivery of molecules into a biological system, such as cells. Theconjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosmall molecules, lipids, nucleosides, nucleotides, nucleic acids,polynucleotides, oligonucleotides, antibodies, toxins, negativelycharged polymers and other polymers, for example proteins, peptides,hormones, carbohydrates, or polyamines, across cellular membranes. Ingeneral, the transporters described are designed to be used eitherindividually or as part of a multi-component system, with or withoutdegradable linkers. The compounds of the invention generally shown inthe Formulae below are expected to improve delivery of molecules into anumber of cell types originating from different tissues, in the presenceor absence of serum.

The present invention features a compound having the Formula 1:

wherein each R₁, R₃, R₄, R₅, R₆, R₇ and R₈ is independently hydrogen,alkyl substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a phosphorus containing group, nucleoside,nucleotide, small molecule, nucleic acid, polynucleotide, oroligonucleotide such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, siNA or a portion thereof, or a solid supportcomprising a linker.

The present invention features a compound having the Formula 2:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, nucleic acid, polynucleotide, or oligonucleotide such as anenzymatic nucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, siNA or a portionthereof, or a solid support comprising a linker.

The present invention features a compound having the Formula 3:

wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, or nucleic acid, polynucleotide, or oligonucleotide such as anenzymatic nucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, siNA or a portionthereof.

The present invention features a compound having the Formula 4:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₂ is a phosphoruscontaining group, nucleoside, nucleotide, small molecule, nucleic acid,polynucleotide, or oligonucleotide such as an enzymatic nucleic acid,allozyme, antisense nucleic acid, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, siNA or a portion thereof, or a solidsupport comprising a linker, and R₁₃ is an amino acid side chain.

The present invention features a compound having the Formula 5:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆, R₇ and R₈ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

The present invention features a compound having the Formula 6:

wherein each R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, R₂ isa phosphorus containing group, nucleoside, nucleotide, small molecule,nucleic acid, polynucleotide, or oligonucleotide such as an enzymaticnucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy,aptamer or triplex forming oligonucleotide, siNA or a portion thereof,or a solid support comprising a linker, each “n” is independently aninteger from 0 to about 200, and L is a degradable linker.

The present invention features a compound having the Formula 7:

wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a phosphorus containing group, nucleoside, nucleotide, smallmolecule, nucleic acid, polynucleotide, or oligonucleotide such as anenzymatic nucleic acid, allozyme, antisense nucleic acid, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, siNA or a portionthereof, or a solid support comprising a linker.

The present invention features a compound having the Formula 8:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

The present invention features a method for synthesizing a compoundhaving Formula 5:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group,comprising: coupling a bis-hydroxy aminoalkyl derivative, for exampleD-threoninol, with a N-protected aminoalkanoic acid to yield a compoundof Formula 9;

wherein R₁₁ is an amino protecting group, R₁₂ is a straight or branchedchain alkyl, substituted alkyl, aryl, or substituted aryl, and each “n”is independently an integer from 0 to about 200; introducing primaryhydroxy protection R₁ followed by amino deprotection of R₁₁ to yield acompound of Formula 10;

wherein R₁ is a protecting group, R₁₂ is a straight or branched chainalkyl, substituted alkyl, aryl, or substituted aryl, and each “n” isindependently an integer from 0 to about 200; coupling the deprotectedamine of Formula 10 with a protected amino acid, for example glutamicacid, to yield a compound of Formula 11;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each “n” is independently an integer from 0 to about 200, R₁₁ is anamino protecting group, and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl; deprotecting the amine R₁₁of the conjugated glutamic acid of Formula XI to yield a compound ofFormula 12;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each “n” is independently an integer from 0 to about 200, R₁ is an aminoprotecting group, and R₁₂ is a straight or branched chain alkyl,substituted alkyl, aryl, or substituted aryl; coupling the deprotectedamine of Formula 12 with an amino protected pteroic acid to yield acompound of Formula 13;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each “n” is independently aninteger from 0 to about 200; and introducing a phosphorus containinggroup at the secondary hydroxyl of Formula 13 to yield a compound ofFormula 5.

The present invention features a method for synthesizing a compoundhaving Formula 8:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, each R₉ and R₁₀ is independently a nitrogen containing group,cyanoalkoxy, alkoxy, aryloxy, or alkyl group, and R₁₂ is a straight orbranched chain alkyl, substituted alkyl, aryl, or substituted aryl,comprising; coupling a bis-hydroxy aminoalkyl derivative, for exampleD-threoninol, with a protected amino acid, for example glutamic acid, toyield a compound of Formula 14;

wherein R₁₁ is an amino protecting group, each “n” is independently aninteger from 0 to about 200, R₄ is independently a protecting group, andR₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl; introducing primary hydroxy protection R₁ followed byamino deprotection of R₁₁ of Formula 14 to yield a compound of Formula15;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and each “n” is independently an integer from 0 toabout 200; coupling the deprotected amine of Formula 15 with an aminoprotected pteroic acid to yield a compound of Formula 16;

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl, and each “n” is independently aninteger from 0 to about 200; and introducing a phosphorus containinggroup at the secondary hydroxyl of Formula 16 to yield a compound ofFormula 8.

In one embodiment, R₂ of a compound of the invention comprises aphosphorus containing group.

In another embodiment, R₂ of a compound of the invention comprises anucleoside, for example, a nucleoside with beneficial activity such asanticancer or antiviral activity.

In yet another embodiment, R₂ of a compound of the invention comprises anucleotide, for example, a nucleotide with beneficial activity such asanticancer or antiviral activity.

In a further embodiment, R₂ of a compound of the invention comprises asmall molecule, for example, a small molecule with beneficial activitysuch as anticancer or antiviral activity.

In another embodiment, R₂ of a compound of the invention comprises anucleic acid, polynucleotide, or oligonucleotide, for example, a nucleicacid, polynucleotide, or oligonucleotide with beneficial activity suchas anticancer or antiviral activity. Non-limiting examples of nucleicacid, polynucleotide, and oligonucleotide compounds include enzymaticnucleic acid molecules, antisense molecules, aptamers, triplex formingoligonucleotides, decoys, 2,5-A chimera molecules, and siNA or a portionthereof.

In one embodiment, R₂ of a compound of the invention comprises a solidsupport comprising a linker.

In another embodiment, a nucleoside (R₂) of the invention comprises anucleoside with anticancer activity.

In another embodiment, a nucleoside (R₂) of the invention comprises anucleoside with antiviral activity.

In another embodiment, the nucleoside (R₂) of the invention comprisesfludarabine, lamivudine (3TC), 5-fluoro uridine, AZT, ara-adenosine,ara-adenosine monophosphate, a dideoxy nucleoside analog,carbodeoxyguanosine, ribavirin, fialuridine, lobucavir, a pyrophosphatenucleoside analog, an acyclic nucleoside analog, acyclovir,gangciclovir, penciclovir, famciclovir, an L-nucleoside analog, FTC,L-FMAU, L-ddC, L-FddC, L-d4C, L-Fd4C, an L-dideoxypurine nucleosideanalog, cytallene, bis-POM PMEA (GS-840), BMS-200,475, carbovir orabacavir.

In one embodiment, R₁₃ of a compound of the invention comprises analkylamino or an alkoxy group, for example, —CH₂O— or —CH(CH₂)CH₂O—.

In another embodiment, R₁₂ of a compound of the invention is analkylhyrdroxyl, for example, —(CH₂)_(n)OH, where n comprises an integerfrom about Ito about 10.

In another embodiment, L of Formula 6 of the invention comprises serine,threonine, or a photolabile linkage.

In one embodiment, R₉ of a compound of the invention comprises aphosphorus protecting group, for example —OCH₂CH₂CN (oxyethylcyano).

In one embodiment, R₁₀ of a compound of the invention comprises anitrogen containing group, for example, —N(R₁₄) wherein R₁₄ is astraight or branched chain alkyl having from about 1 to about 10carbons.

In another embodiment, R₁₀ of a compound of the invention comprises aheterocycloalkyl or heterocycloalkenyl ring containing from about 4 toabout 7 atoms, and having from about 1 to about 3 heteroatoms comprisingoxygen, nitrogen, or sulfur.

In another embodiment, R₁ of a compound of the invention comprises anacid labile protecting group, such as a trityl or substituted tritylgroup, for example, a dimethoxytrityl or mono-methoxytrityl group.

In another embodiment, R₄ of a compound of the invention comprises atert-butyl, Fm (fluorenyl-methoxy), or allyl group.

In one embodiment, R₆ of a compound of the invention comprises a TFA(trifluoracetyl) group.

In another embodiment, R₃, R₅, R₇ and R₈ of a compound of the inventionare independently hydrogen.

In one embodiment, R₇ of a compound of the invention is independentlyisobutyryl, dimethylformamide, or hydrogen.

In another embodiment, R₁₂ of a compound of the invention comprises amethyl group or ethyl group.

In one embodiment, a nucleic acid of the invention comprises a siNAmolecule or a portion thereof.

In one embodiment, a nucleic acid of the invention comprises anenzymatic nucleic acid, for example a hammerhead, Inozyme, DNAzyme,G-cleaver, Zinzyme, Amberzyme, or allozyme or a portion thereof.

In another embodiment, a nucleic acid of the invention comprises anantisense nucleic acid, 2-5A nucleic acid chimera, or decoy nucleic acidor a portion thereof.

In another embodiment, the solid support having a linker of theinvention comprises a structure of Formula 17:

wherein SS is a solid support, and each “n” is independently an integerfrom about 1 to about 200.

In another embodiment, the solid support of the instant invention iscontrolled pore glass (CPG) or polystyrene, and can be used in thesynthesis of a nucleic acid, polynucleotide, or oligonucleotide or theinvention, such as an enzymatic nucleic acid, allozyme, antisensenucleic acid, 2,5-A chimera, decoy, aptamer, triplex formingoligonucleotide, siNA or a portion thereof.

In one embodiment, the invention features a pharmaceutical compositioncomprising a compound of the invention and a pharmaceutically acceptablecarrier.

In another embodiment, the invention features a method of treating acancer patient, comprising contacting cells of the patient with apharmaceutical composition of the invention under conditions suitablefor the treatment. This treatment can comprise the use of one or moreother drug therapies under conditions suitable for the treatment. Thecancers contemplated by the instant invention include but are notlimited to breast cancer, lung cancer, colorectal cancer, brain cancer,esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer,cervical cancer, head and neck cancer, ovarian cancer, melanoma,lymphoma, glioma, or multidrug resistant cancers.

In one embodiment, the invention features a method of treating a patientinfected with a virus, comprising contacting cells of the patient with apharmaceutical composition of the invention, under conditions suitablefor the treatment. This treatment can comprise the use of one or moreother drug therapies under conditions suitable for the treatment. Theviruses contemplated by the instant invention include but are notlimited to HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza,rhinovirus, west nile virus, Ebola virus, foot and mouth virus, andpapilloma virus.

In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule or other target molecule in asample, for example, a gene in a cancer cell, comprising a compound ofthe instant invention.

In one embodiment, the invention features a kit for detecting thepresence of a nucleic acid molecule, or other target molecule in asample, for example, a gene in a virus-infected cell, comprising acompound of the instant invention.

In another embodiment, the invention features a compound of the instantinvention comprising a modified phosphate group, for example, aphosphoramidite, phosphodiester, phosphoramidate, phosphorothioate,phosphorodithioate, alkylphosphonate, arylphosphonate, monophosphate,diphosphate, triphosphate, or pyrophosphate.

In one embodiment, the invention features a method for synthesizing acompound having Formula 18:

wherein each R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, comprising: reacting folic acid with acarboxypeptidase to yield a compound of Formula 19;

introducing a protecting group R₆ on the secondary amine of Formula 19to yield a compound of Formula 20;

wherein R₆ is a nitrogen protecting group; and introducing a protectinggroup R₇ on the primary amine of Formula 20 to yield a compound ofFormula 18.

In another embodiment, the amino protected pteroic acid of the inventionis a compound of Formula 18.

In one embodiment, the invention encompasses a compound of Formula 1having Formula 21:

wherein each “n” is independently an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of Formula 7having Formula 22:

wherein each “n” is independently an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of Formula 4having Formula 23:

wherein “n” is an integer from 0 to about 200.

In another embodiment, the invention encompasses a compound of Formula 4having Formula 24:

wherein “n” is an integer from 0 to about 200.

In another embodiment, the invention features a compound having Formula25:

wherein each R₅ and R₇ is independently hydrogen, alkyl or a nitrogenprotecting group, each R₁₅, R₁₆, R₁₇, and R₁₈ is independently O, S,alkyl, substituted alkyl, aryl, substituted aryl, or halogen, X₁ is—CH(X_(1′)) or a group of Formula 38:

wherein R₄ is a protecting group and “n” is an integer from 0 to about200;X_(1′) is the protected or unprotected side chain of a naturallyoccurring or non-naturally-occurring amino acid, X₂ is amide, alkyl, orcarbonyl containing linker or a bond, and X₃ is a degradable linkerwhich is optionally absent.

In another embodiment, the X₃ group of Formula 25 comprises a group ofFormula 26:

wherein R₄ is hydrogen or a protecting group, “n” is an integer from 0to about 200 and R₁₂ is a straight or branched chain alkyl, substitutedalkyl, aryl, or substituted aryl.

In yet another embodiment, R₄ of Formula 26 is hydrogen and R₁₂ ismethyl or hydrogen.

In still another embodiment, the invention features a compound havingFormula 27:

wherein “n” is an integer from about 0 to about 20, R₄ is H or acationic salt, and R₂₄ is a sulfur containing leaving group, for examplea group comprising:

In another embodiment, the invention features a method for synthesizinga compound having Formula 27 comprising:

(a) selective tritylation of the thiol of cysteamine under conditionssuitable to yield a compound having Formula 28:

wherein “n” is an integer from about 0 to about 20 and R₁₉ is a thiolprotecting group;(b) peptide coupling of the product of (a) with a compound havingFormula 29:

wherein R₂₀ is a carboxylic acid protecting group and R₂₁ is an aminoprotecting group, under conditions suitable to yield a compound havingFormula 30:

wherein “n” is an integer from about 0 to about 20, R₁₉ is a thiolprotecting group, R₂₀ is a carboxylic acid protecting group and R₂₁ isan amino protecting group;(c) removing the amino protecting group R₂₁ of the product of (b) underconditions suitable to yield a compound having Formula 31:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b);(d) condensation of the product of (c) with a compound having Formula32:

wherein R₂₂ is an amino protecting group, under conditions suitable toyield a compound having Formula 33:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b) and R₂₂ is as described in (d);(e) selective cleavage of R₂₂ from the product of (d) under conditionssuitable to yield a compound having Formula 34:

wherein “n” is an integer from about 0 to about 20 and R₁₉ and R₂₀ areas described in (b);(f) coupling the product of (e) with a compound having Formula 35:

wherein R₂₃ is an amino protecting group under conditions suitable toyield a compound having Formula 36:

wherein R₂₃ is an amino protecting group, “n” is an integer from about 0to about 20 and R₁₉ and R₂₀ are as described in (b);(g) deprotecting the product of (f) under conditions suitable to yield acompound having Formula 37.

wherein “n” is an integer from about 0 to about 20; and(h) introducing a disulphide-based leaving group to the product of (g)under conditions suitable to yield a compound having Formula 27.

In one embodiment, the invention features a compound having Formula 39:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide such as an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, and P is a phosphorus containing group.In another embodiment, X comprises a siNA molecule or a portion thereof.

In another embodiment, the invention features a method for synthesizinga compound having Formula 39, comprising:

(a) coupling a thiol containing linker to a nucleic acid, polynucleotideor oligonucleotide under conditions suitable to yield a compound havingFormula 40:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide, and P is a phosphorus containinggroup; and(b) coupling the product of (a) with a compound having Formula 37 underconditions suitable to yield a compound having Formula 39.

In another embodiment, the thiol containing linker of the invention is acompound having Formula 41:

wherein “n” is an integer from about 0 to about 20, P is a phosphoruscontaining group, for example a phosphine, phosphite, or phosphate, andR₂₄ is any alkyl, substituted alkyl, alkoxy, aryl, substituted aryl,alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group withor without additional protecting groups.

In another embodiment, the conditions suitable to yield a compoundhaving Formula 40 comprises reduction, for example using dithiothreitol(DTT) or any equivalent disulphide reducing agent, of the disulfide bondof a compound having Formula 42:

wherein “n” is an integer from about 0 to about 20, X is a nucleic acid,polynucleotide, or oligonucleotide, P is a phosphorus containing group,and R₂₄ is any alkyl, substituted alkyl, alkoxy, aryl, substituted aryl,alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group withor without additional protecting groups. In another embodiment, Xcomprises a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 43:

wherein X comprises a biologically active molecule; W comprises adegradable nucleic acid linker; Y comprises a linker molecule or aminoacid that can be present or absent; Z comprises H, OH, O-alkyl, SH,S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, orlabel; n is an integer from about 1 to about 100; and N′ is an integerfrom about 1 to about 20. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, W is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula44:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; n is aninteger from about 1 to about 50, and PEG represents a compound havingFormula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100. In another embodiment, X comprises a siNA molecule or a portionthereof. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula46:

wherein X comprises a biologically active molecule; each W independentlycomprises linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule or chemical linkage that can bepresent or absent; and PEG represents a compound having Formula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100. In another embodiment, X comprises a siNA molecule or a portionthereof. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In one embodiment, the invention features a compound having Formula 47:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be the same ordifferent and can be present or absent, Y comprises a linker moleculethat can be present or absent; each Q independently comprises ahydrophobic group or phospholipid; each R1, R2, R3, and R4 independentlycomprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl,S-alkylcyano, N or substituted N, and n is an integer from about 1 toabout 10. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula48:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and B represents a lipophilic group, for example asaturated or unsaturated linear, branched, or cyclic alkyl group,cholesterol, or a derivative thereof. In another embodiment, X comprisesa siNA molecule or a portion thereof. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula49:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and Brepresents a lipophilic group, for example a saturated or unsaturatedlinear, branched, or cyclic alkyl group, cholesterol, or a derivativethereof. In another embodiment, X comprises a siNA molecule or a portionthereof. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula50:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule or chemical linkage that can be present or absent; andeach Q independently comprises a hydrophobic group or phospholipid. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the invention features a compound having Formula 51:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; Y comprisesa linker molecule or amino acid that can be present or absent; Zcomprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,substituted aryl, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, or label; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and n is aninteger from about 1 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula52:

wherein X comprises a biologically active molecule; Y comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,R3, R4, and R5 independently comprises O, OH, H, alkyl, alkylhalo,O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N; Zcomprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,substituted aryl, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, or label; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, n is an integerfrom about 1 to about 20; and N′ is an integer from about 1 to about 20.In another embodiment, X comprises a siNA molecule or a portion thereof.In another embodiment, Y is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula53:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, N, S,alkyl, or substituted N; each R2 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; each R3 independently comprises N or O—N,each R4 independently comprises O, CH₂, S, sulfone, or sulfoxy; Xcomprises H, a removable protecting group, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymatic nucleicacid, siNA or a portion thereof, amino acid, peptide, protein, lipid,phospholipid, or label; W comprises a linker molecule or chemicallinkage that can be present or absent; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,each n is independently an integer from about 1 to about 50; and N′ isan integer from about 1 to about 10. In another embodiment, X comprisesa siNA molecule or a portion thereof. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula54:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, siNA or a portion thereof,amino acid, peptide, protein, lipid, phospholipid, or label; W comprisesa linker molecule or chemical linkage that can be present or absent; andSG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 55:

wherein each R1 independently comprises O, N, S, alkyl, or substitutedN; each R2 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylhalo, S, N, substituted N, or a phosphorus containing group; eachR3 independently comprises H, OH, alkyl, substituted alkyl, or halo; Xcomprises H, a removable protecting group, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide such as anenzymatic nucleic acid, allozyme, antisense nucleic acid, siNA or aportion thereof, 2,5-A chimera, decoy, aptamer or triplex formingoligonucleotide, amino acid, peptide, protein, lipid, phospholipid,biologically active molecule or label; W comprises a linker molecule orchemical linkage that can be present or absent; SG comprises a sugar,for example galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, each n is independently an integer from about 1 to about 50;and N′ is an integer from about 1 to about 100. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula56:

wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide such as an enzymatic nucleicacid, allozyme, antisense nucleic acid, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, siNA or a portion thereof, amino acid,peptide, protein, lipid, phospholipid, biologically active molecule orlabel; W comprises a linker molecule or chemical linkage that can bepresent or absent; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and each n isindependently an integer from about 0 to about 20. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula57:

wherein R1 can include the groups:

and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,and n is an integer from about 1 to about 20.

In one embodiment, compounds having Formula 52, 53, 54, 55, 56, and 57are featured wherein each nitrogen adjacent to a carbonyl canindependently be substituted for a carbonyl adjacent to a nitrogen oreach carbonyl adjacent to a nitrogen can be substituted for a nitrogenadjacent to a carbonyl.

In another embodiment, the invention features a compound having Formula58:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; Y comprisesa linker molecule or amino acid that can be present or absent; Vcomprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each n is independentlyan integer from about 1 to about 50; and N′ is an integer from about 1to about 100. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula59:

wherein each R1 independently comprises O, S, N, substituted N, or aphosphorus containing group; each R2 independently comprises O, S, or N;X comprises H, amino, substituted amino, nucleotide, nucleoside, nucleicacid, oligonucleotide, or enzymatic nucleic acid or other biologicallyactive molecule; n is an integer from about 1 to about 50, Q comprises Hor a removable protecting group which can be optionally absent, each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, and V comprises a signal protein or peptide, forexample Human serum albumin protein, Antennapedia peptide, Kaposifibroblast growth factor peptide, Caiman crocodylus Ig(5) light chainpeptide, HIV envelope glycoprotein gp41 peptide, HIV-1 Tat peptide,Influenza hemagglutinin envelope glycoprotein peptide, or transportan Apeptide, or a compound having Formula 45

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide such as an enzymatic nucleicacid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy,aptamer or triplex forming oligonucleotide, amino acid, peptide,protein, lipid, phospholipid, or label; and n is an integer from about 1to about 100. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula60:

wherein R1 can include the groups:

and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; n is an integer from about 1 toabout 50; and R8 is a nitrogen protecting group, for example aphthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group.

In another embodiment, the invention features a compound having Formula61:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be the same ordifferent and can be present or absent, Y comprises a linker moleculethat can be present or absent; each 5 independently comprises a signalprotein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and n is aninteger from about 1 to about 10. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula62:

wherein X comprises a biologically active molecule; each 5 independentlycomprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; W comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,and R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and each nis independently an integer from about 1 to about 10. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula63:

wherein X comprises a biologically active molecule; V comprises a signalprotein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; W comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, R4represents an ester, amide, or protecting group, and each n isindependently an integer from about 1 to about 10. In anotherembodiment, X comprises a siNA molecule or a portion thereof. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula64:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, A comprises a nitrogen containing group, and B comprisesa lipophilic group. In another embodiment, X comprises a siNA moleculeor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula65:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, RV comprises the lipid or phospholipid component of anyof Formulae 47-50, and R6 comprises a nitrogen containing group. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula92:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, biologically active molecule or label; W comprisesa linker molecule or chemical linkage that can be present or absent; R2comprises O, NH, S, CO, COO, ON═C, or alkyl; R3 comprises alkyl, akloxy,or an aminoacyl side chain; and SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula86:

wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid, aminoacid, peptide, protein, lipid, phospholipid, biologically activemolecule or label; W comprises a linker molecule or chemical linkagethat can be present or absent; R3 comprises O, NH, S, CO, COO, ON═C, oralkyl; R4 comprises alkyl, akloxy, or an aminoacyl side chain; and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, and each n is independently aninteger from about 0 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula87:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent; and Y comprises abiologically active molecule, for example an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, peptide, protein, or antibody; R1comprises H, alkyl, or substituted alkyl. In another embodiment, Xcomprises a siNA molecule or a portion thereof. In another embodiment, Wis selected from the group consisting of amide, phosphate, phosphateester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula88:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent, and Y comprises abiologically active molecule, for example an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, peptide, protein, or antibody. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula99:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine or branched derivative thereof,glucose, mannose, fructose, or fucose and the respective D or L, alphaor beta isomers. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula100:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine or branched derivative thereof, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the SG component of any compound having Formulae 99or 100 comprises a compound having Formula 101:

wherein Y comprises a linker molecule or chemical linkage that can bepresent or absent and each R7 independently is hydrogen or an acylgroup, for example an acetyl group.

In one embodiment, the W-SG component of a compound having Formulae 99comprises a compound having Formula 102:

wherein R2 comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo,S, N, substituted N, a protecting group, or another compound havingFormula 102; R1 independently H, OH, alkyl, substituted alkyl, or haloand each R7 independently is hydrogen or an acyl group, for example anacetyl group, and R3 comprises O or R3 in Formula 99, and n is aninteger from about 1 to about 20.

In one embodiment, the W-SG component of a compound having Formulae 99comprises a compound having Formula 103:

wherein R1 comprises H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N,substituted N, a protecting group, or another compound having Formula103; each R7 independently is hydrogen or an acyl group, for example anacetyl group, and R3 comprises H or R3 in Formula 99, and each n isindependently an integer from about 1 to about 20.

In one embodiment, the invention features a compound having Formula 104:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,and each n is independently an integer from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In one embodiment, the invention features a compound having Formula 105:

wherein X comprises a nucleotide, polynucleotide, or oligonucleotide ora portion thereof, R2 comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylhalo, S, N, substituted N, a protecting group, or a nucleotide,polynucleotide, or oligonucleotide or a portion thereof; R1independently H, OH, alkyl, substituted alkyl, or halo and each R7independently is hydrogen or an acyl group, for example an acetyl group,and n is an integer from about 1 to about 20. In another embodiment, Xcomprises a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 106:

wherein X comprises a nucleotide, polynucleotide, or oligonucleotide ora portion thereof, R1 comprises H, OH, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, label, or a portion thereof, orOR5 where R5 a removable protecting group, each R7 independently ishydrogen or an acyl group, for example an acetyl group, and each n isindependently an integer from about 1 to about 20. In anotherembodiment, X comprises a siNA molecule or a portion thereof.

In another embodiment, the invention features a compound having Formula107:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and Cholesterol comprises cholesterol or an analog,derivative, or metabolite thereof. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula108:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, andCholesterol comprises cholesterol or an analog, derivative, ormetabolite thereof. In another embodiment, X comprises a siNA moleculeor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In one embodiment, the W-Cholesterol component of a compound havingFormula 107 comprises a compound having Formula 109:

wherein R3 comprises R3 as described in Formula 107, and n isindependently an integer from about 1 to about 20.

In one embodiment, the invention features a compound having Formula 110:

wherein R4 comprises O, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S,S-alkyl, S-alkylcyano, N or substituted N, each n is independently aninteger from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In one embodiment, the invention features a compound having Formula 111:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and n is aninteger from about 1 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 112:

wherein n is an integer from about 1 to about 20. In another embodiment,a compound having Formula 112 is used to generate a compound havingFormula 111 via NHS ester mediated coupling with a biologically activemolecule, such as a siNA molecule or a portion thereof. In anon-limiting example, the NHS ester coupling can be effectuated viaattachment to a free amine present in the siNA molecule, such as anamino linker molecule present on a nucleic acid sugar (e.g. 2′-aminolinker) or base (e.g., C5 alkyl amine linker) component of the siNAmolecule.

In one embodiment, the invention features a compound having Formula 113:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 113 is used to generatea compound having Formula 111 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a compound having Formula 114:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and n is aninteger from about 1 to about 20. In another embodiment, X comprises asiNA molecule or a portion thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 115:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, R3 comprisesH, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,label, or a portion thereof, or OR5 where R5 a removable protectinggroup, and each n is independently an integer from about 1 to about 20.In another embodiment, X comprises a siNA molecule or a portion thereof.In another embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the invention features a compound having Formula 116:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 116 is used to generatea compound having Formula 114 or 115 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

In one embodiment, the invention features a compound having Formula 117:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 117 is used to generatea compound having Formula 105 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a compound having Formula 118:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, R3 comprisesH, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,label, or a portion thereof, or OR5 where R5 a removable protectinggroup, each R7 independently is hydrogen or an acyl group, for examplean acetyl group, and each n is independently an integer from about 1 toabout 20. In another embodiment, X comprises a siNA molecule or aportion thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 119:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, each R7independently is hydrogen or an acyl group, for example an acetyl group,and each n is independently an integer from about 1 to about 20. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the invention features a compound having Formula 120:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 120 is used to generatea compound having Formula 118 or 119 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

In one embodiment, the invention features a compound having Formula 121:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, each R7independently is hydrogen or an acyl group, for example an acetyl group,and each n is independently an integer from about 1 to about 20. Inanother embodiment, X comprises a siNA molecule or a portion thereof. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the invention features a compound having Formula 122:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently is hydrogen or an acyl group, for example an acetyl group,each n is independently an integer from about 1 to about 20, andwherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 122 is used to generatea compound having Formula 121 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a method for the synthesis ofa compound having Formula 48:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N; and each B independently represents a lipophilic group,for example a saturated or unsaturated linear, branched, or cyclic alkylgroup, comprising: (a) introducing a compound having Formula 66:

wherein R1 is defined as in Formula 48 and can include the groups:

and wherein R2 is defined as in Formula 48 and can include the groups:

and wherein each R5 independently comprises O, N, or S and each R6independently comprises a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl group, to a compoundhaving Formula 67:X—W—Y  67wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 68:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; and each R1, R2, R3,and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N comprising,each R5 independently comprises O, S, or N; and each R6 is independentlya removable protecting group, for example a trityl, monomethoxytrityl,or dimethoxytrityl group; (b) removing R6 from the compound havingFormula 26 and (c) introducing a compound having Formula 69:

wherein R1 is defined as in Formula 48 and can include the groups:

and wherein R2 is defined as in Formula 48 and can include the groups:

and wherein W and B are defined as in Formula 48, to the compound havingFormula 68 under conditions suitable for the formation of a compoundhaving Formula 48.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 49:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N; each R5independently comprises O, S, or N; and each B independently comprises alipophilic group, for example a saturated or unsaturated linear,branched, or cyclic alkyl group, comprising: (a) coupling a compoundhaving Formula 70:

wherein R1 is defined as in Formula 49 and can include the groups:

and wherein R2 is defined as in Formula 49 and can include the groups:

and wherein each R5 independently comprises O, S, or N, and wherein eachB independently comprises a lipophilic group, for example a saturated orunsaturated linear, branched, or cyclic alkyl group, with a compoundhaving Formula 67:X—W—Y  67wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 49.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 52:

wherein X comprises a biologically active molecule; Y comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,R3, and R4 independently comprises 0, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N; Z comprisesH, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl, substitutedaryl, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, orlabel; SG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, n is an integer from about 1to about 20; and N′ is an integer from about 1 to about 20, comprising:(a) coupling a compound having Formula 71:

wherein R1, R2, R3, R5, SG, and n is as defined in Formula 52, andwherein R1 can include the groups:

and wherein R2 can include the groups:

and R6 comprises a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl group; with a compound havingFormula 72:X—Y  72wherein X comprises a biologically active molecule and Y comprises alinker molecule that can be present or absent, under conditions suitablefor the formation of a compound having Formula 95:

(b) removing R6 from the compound having Formula 95 and (c) optionallycoupling a nucleotide, nucleoside, nucleic acid, oligonucleotide, aminoacid, peptide, protein, lipid, phospholipid, or label, or optionally;coupling a compound having Formula 71 under and optionally repeating (b)and (c) under conditions suitable for the formation of a compound havingFormula 52.

In another embodiment, the invention features a method for synthesizinga compound having Formula 53:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, N, S,alkyl, or substituted N; each R2 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; each R3 independently comprises N or O—N,each R4 independently comprises O, CH₂, S, sulfone, or sulfoxy; Xcomprises H, a removable protecting group, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymatic nucleicacid, amino acid, peptide, protein, lipid, phospholipid, or label; Wcomprises a linker molecule or chemical linkage that can be present orabsent; SG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, each n is independently aninteger from about 1 to about 50; and N′ is an integer from about 1 toabout 10, comprising: coupling a compound having Formula 73:

wherein R1, R2, R3, R4, X, W, B, N′ and n are as defined in Formula 53,with a sugar, for example a compound having Formula 74:

wherein Y comprises a linker molecule or chemical linkage that can bepresent or absent; L represents a reactive chemical group, for example aNHS ester, and each R7 independently is hydrogen or an acyl group, forexample an acetyl group; under conditions suitable for the formation ofa compound having Formula 53.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 54:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, biologically active molecule or label; W comprisesa linker molecule or chemical linkage that can be present or absent; SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, comprising (a) coupling acompound having Formula 75:

wherein R1, R2, R3, R4, X, W, and B are as defined in Formula 53, with asugar, for example a compound having Formula 74.

wherein Y comprises a C11 alkyl linker molecule; L represents a reactivechemical group, for example a NHS ester, and each R7 independently ishydrogen or an acyl group, for example an acetyl group; under conditionssuitable for the formation of a compound having Formula 54.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 55:

wherein each R1 independently comprises O, N, S, alkyl, or substitutedN; each R2 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylhalo, S, N, substituted N, or a phosphorus containing group; eachR3 independently comprises H, OH, alkyl, substituted alkyl, or halo; Xcomprises H, a removable protecting group, nucleotide, nucleoside,nucleic acid, oligonucleotide, or enzymatic nucleic acid or biologicallyactive molecule; W comprises a linker molecule or chemical linkage thatcan be present or absent; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, each n isindependently an integer from about 1 to about 50; and N′ is an integerfrom about 1 to about 100, comprising: (a) coupling a compound havingFormula 76:

wherein R1 can include the groups:

and wherein R2 can include the groups:

and wherein each R3 independently comprises H, OH, alkyl, substitutedalkyl, or halo; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and n is aninteger from about 1 to about 20, to a compound X—W, wherein X comprisesa nucleotide, nucleoside, nucleic acid, oligonucleotide, enzymaticnucleic acid, amino acid, peptide, protein, lipid, phospholipid,biologically active molecule or label, and W comprises a linker moleculeor chemical linkage that can be present or absent; and (b) optionallyrepeating step (a) under conditions suitable for the formation of acompound having Formula 55.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 56:

wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, enzymatic nucleic acid, aminoacid, peptide, protein, lipid, phospholipid, biologically activemolecule or label; W comprises a linker molecule or chemical linkagethat can be present or absent; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,and each n is independently an integer from about 0 to about 20,comprising: (a) coupling a compound having Formula 77:

wherein each R1, X, W, and n are as defined in Formula 56, to a sugar,for example a compound having Formula 74:

wherein Y comprises an alkyl linker molecule of length n, where n is aninteger from about 1 to about 20; L represents a reactive chemicalgroup, for example a NHS ester, and each R7 independently is hydrogen oran acyl group, for example an acetyl group; and (b) optionally couplingX—W, wherein X comprises a removable protecting group, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, or label and W comprises a linker molecule orchemical linkage that can be present or absent, under conditionssuitable for the formation of a compound having Formula 56.

In another embodiment, the invention features method for synthesizing acompound having Formula 57:

wherein R1 can include the groups:

and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,and n is an integer from about 1 to about 20, comprising: (a) coupling acompound having Formula 77:

wherein R1 and X comprise H, to a sugar, for example a compound havingFormula 74:

wherein Y comprises an alkyl linker molecule of length n, where n is aninteger from about 1 to about 20; L represents a reactive chemicalgroup, for example a NHS ester, and each R7 independently is hydrogen oran acyl group, for example an acetyl group; and (b) introducing a tritylgroup, for example a dimethoxytrityl, monomethoxytrityl, or trityl groupto the primary hydroxyl of the product of (a) and (c) introducing aphosphorus containing group having Formula 78:

wherein R1 can include the groups:

and wherein each R2 and R3 independently can include the groups:

to the secondary hydroxyl of the product of (b) under conditionssuitable for the formation of a compound having Formula 57.

In another embodiment, the invention features a method for synthesizinga compound having Formula 60:

wherein R1 can include the groups:

and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; n is an integer from about 1 toabout 50; and R8 is a nitrogen protecting group, for example aphthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group,comprising: (a) introducing carboxy protection to a compound havingFormula 79:

wherein n is an integer from about 1 to about 50, under conditionssuitable for the formation of a compound having Formula 80:

wherein n is an integer from about 1 to about 50 and R7 is a carboxylicacid protecting group, for example a benzyl group; (b) introducing anitrogen containing group to the product of (a) under conditionssuitable for the formation of a compound having Formula 81:

wherein n and R7 are as defined in Formula 80 and R8 is a nitrogenprotecting group, for example a phthaloyl, trifluoroacetyl, FMOC, ormonomethoxytrityl group; (c) removing the carboxylic acid protectinggroup from the product of (b) and introducing aminopropanediol underconditions suitable for the formation of a compound having Formula 82:

wherein n and R8 are as defined in Formula 81; (d) introducing aremovable protecting group, for example a trityl, monomethoxytrityl, ordimethoxytrityl to the product of (c) under conditions suitable for theformation of a compound having Formula 83:

wherein Tr, n and R8 are as defined in Formula 60; and (e) introducing aphosphorus containing group having Formula 78:

wherein R1 can include the groups:

and wherein each R2 and R3 independently can include the groups:

to the product of (d) under conditions suitable for the formation of acompound having Formula 60.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 59:

wherein each R1 independently comprises O, S, N, substituted N, or aphosphorus containing group; each R2 independently comprises O, S, or N;X comprises H, amino, substituted amino, nucleotide, nucleoside, nucleicacid, oligonucleotide, such as an enzymatic nucleic acid, allozyme,antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer or triplexforming oligonucleotide or other biologically active molecule or aportion thereof; n is an integer from about 1 to about 50, Q comprises Hor a removable protecting group which can be optionally absent, each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, and V comprises a protein or peptide, for exampleHuman serum albumin protein, Antennapedia peptide, Kaposi fibroblastgrowth factor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide,or a compound having Formula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100, comprising: (a) removing R8 from a compound having Formula 84:

wherein Q, X, W, R1, R2, and n are as defined in Formula 59 and R8 is anitrogen protecting group, for example a phthaloyl, trifluoroacetyl,FMOC, or monomethoxytrityl group, under conditions suitable for theformation of a compound having Formula 85:

wherein Q, X, W, R1, R2, and n are as defined in Formula 59; (b)introducing a group V to the product of (a) via the formation of anoxime linkage, wherein V comprises a protein or peptide, for exampleHuman serum albumin protein, Antennapedia peptide, Kaposi fibroblastgrowth factor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide,or a compound having Formula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100, under conditions suitable for the formation of a compound havingFormula 59.

In another embodiment, the invention features a method for synthesizinga compound having Formula 64:

wherein X comprises a biologically active molecule; each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, Y comprises a linker molecule that can be present or absent;each R1, R2, R3, and R4 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, A comprises a nitrogen containing group, and B comprisesa lipophilic group, comprising: (a) introducing a compound havingFormula 66:

wherein R1 is defined as in Formula 64 and can include the groups:

and wherein R2 is defined as in Formula 64 and can include the groups:

and wherein each R5 independently comprises O, N, or S and each R6independently comprises a removable protecting group, for example atrityl, monomethoxytrityl, or dimethoxytrityl group, to a compoundhaving Formula 67:X—W—Y  67wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, and Ycomprises a linker molecule that can be present or absent, underconditions suitable for the formation of a compound having Formula 68:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent, Y comprisesa linker molecule that can be present or absent; and each R1, R2, R3,and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N comprising,each R5 independently comprises O, S, or N; and each R6 is independentlya removable protecting group, for example a trityl, monomethoxytrityl,or dimethoxytrityl group; (b) removing R6 from the compound havingFormula 68 and (c) introducing a compound having Formula 69:

wherein R1 is defined as in Formula 64 and can include the groups:

and wherein R2 is defined as in Formula 64 and can include the groups:

and wherein R3, W and B are defined as in Formula 64; and introducing acompound having Formula 69′:

wherein R1 is defined as in Formula 64 and can include the groups:

and wherein R2 is defined as in Formula 48 and can include the groups:

and wherein R3, W and A are defined as in Formula 64; to the compoundhaving Formula 68 under conditions suitable for the formation of acompound having Formula 64.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 62:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; each 5independently comprises a protein or peptide, for example Human serumalbumin protein, Antennapedia peptide, Kaposi fibroblast growth factorpeptide, Caiman crocodylus Ig(5) light chain peptide, HIV envelopeglycoprotein gp41 peptide, HIV-1 Tat peptide, Influenza hemagglutininenvelope glycoprotein peptide, or transportan A peptide; each R1, R2,and R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and each nis independently an integer from about 1 to about 10, comprising: (a)introducing a compound having Formula 93:

wherein V and n are as defined in Formula 62, to a compound havingFormula 86:

wherein X, W, R1, R2, R3, and n are as defined in Formula 62, underconditions suitable for the formation of a compound having Formula 62.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 63:

wherein X comprises a biologically active molecule; W comprises a linkermolecule or chemical linkage that can be present or absent; V comprisesa protein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each R1, R2, and R3independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, R4represents an ester, amide, or protecting group, and each n isindependently an integer from about 1 to about 10, comprising: (a)introducing a compound having Formula 96:

wherein V and R4 are as defined in Formula 63, to a compound havingFormula 86:

wherein X, W, R1, R2, R3, and n are as defined in Formula 63, underconditions suitable for the formation of a compound having Formula 63.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 87:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent; and Y comprises abiologically active molecule, for example an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, peptide, protein, or antibody; R1comprises H, alkyl, or substituted alkyl, comprising (a) coupling acompound having Formula 89:

wherein Y, W and R are as defined in Formula 87, with a compound havingFormula 90:H₂N—O—X  90wherein X is as defined in Formula 87, under conditions suitable for theformation of a compound having Formula 87, for example by post-syntheticconjugation of a compound having Formula 89 with a compound havingFormula 90, wherein X of compound 90 comprises an enzymatic nucleic acidmolecule and Y of Formula 89 comprises a peptide.

In another embodiment, the invention features a method for the synthesisof a compound having Formula 88:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent, and Y comprises abiologically active molecule, for example an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, peptide, protein, or antibody,comprising (a) coupling a compound having Formula 91:

wherein Y and W are as defined in Formula 88, with a compound havingFormula 90:H₂N—O—X  90wherein X is as defined in Formula 88, under conditions suitable for theformation of a compound having Formula 88, for example by post-syntheticconjugation of a compound having Formula 91 with a compound havingFormula 90, wherein X of compound 90 comprises an enzymatic nucleic acidmolecule and Y of Formula 91 comprises a peptide.

In one embodiment, the invention features a compound having Formula 94,X—Y—W—Y—Z  94wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; each Y independently comprises alinker or chemical linkage that can be present or absent, W comprises abiodegradable nucleic acid linker molecule, and Z comprises abiologically active molecule, for example an enzymatic nucleic acid,allozyme, antisense nucleic acid, siNA, 2,5-A chimera, decoy, aptamer ortriplex forming oligonucleotide, peptide, protein, or antibody.

In another embodiment, W of a compound having Formula 94 of theinvention comprises5′-cytidine-deoxythymidine-3′,5′-deoxythymidine-cytidine-3′,5′-cytidine-deoxyuridine-3′,5′-deoxyuridine-cytidine-3′,5′-uridine-deoxythymidine-3′,or 5′-deoxythymidine-uridine-3′.

In yet another embodiment, W of a compound having Formula 94 of theinvention comprises5′-adenosine-deoxythymidine-3′,5′-deoxythymidine-adenosine-3′,5′-adenosine-deoxyuridine-3′,or 5′-deoxyuridine-adenosine-3′.

In another embodiment, Y of a compound having Formula 94 of theinvention comprises a phosphorus containing linkage, phoshoramidatelinkage, phosphodiester linkage, phosphorothioate linkage, amidelinkage, ester linkage, carbamate linkage, disulfide linkage, oximelinkage, or morpholino linkage.

In another embodiment, compounds having Formula 89 and 91 of theinvention are synthesized by periodate oxidation of an N-terminal Serineor Threonine residue of a peptide or protein.

In one embodiment, X of compounds having Formulae 43, 44, 46-52, 58,61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or121 of the invention comprises a siNA molecule or a portion thereof. Inone embodiment, the siNA molecule can be conjugated at the 5′ end,3′-end, or both 5′ and 3′ ends of the sense strand or region of thesiNA. In one embodiment, the siNA molecule can be conjugated at the3′-end of the antisense strand or region of the siNA with a compound ofthe invention. In one embodiment, both the sense strand and antisensestrands or regions of the siNA molecule are conjugated with a compoundof the invention. In one embodiment, only the sense strand or region ofthe siNA is conjugated with a compound of the invention. In oneembodiment, only the antisense strand or region of the siNA isconjugated with a compound of the invention.

In one embodiment, X of compounds having Formulae 43, 44, 46-52, 58,61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or121 of the invention comprises an enzymatic nucleic acid.

In another embodiment, X of compounds having Formulae 43, 44, 46-52, 58,61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or121 of the invention comprises an antibody. In yet another embodiment, Xof compounds having Formulae 43, 44, 46-52, 58, 61-65, 85-88, 92, 94,95, 99, 100, 105-108, 111, 114, 115, 118, 119, or 121 of the inventioncomprises an interferon.

In another embodiment, X of compounds having Formulae 43, 44, 46-52, 58,61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or121 of the invention comprises an antisense nucleic acid, dsRNA, ssRNA,decoy, triplex oligonucleotide, aptamer, or 2,5-A chimera.

In one embodiment, W and/or Y of compounds having Formulae 43, 44,46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114,115, 118, 119, or 121 of the invention comprises a degradable orcleavable linker, for example a nucleic acid sequence comprisingribonucleotides and/or deoxynucleotides, such as a dimer, trimer, ortetramer. A non limiting example of a nucleic acid cleavable linker isan adenosine-deoxythymidine (A-dT) dimer or a cytidine-deoxythymidine(C-dT) dimer. In yet another embodiment, W and/or V of compounds havingFormulae 43, 44, 48-51, 58, 63-65, 96, 99, 100, 107, 108, 111, 114, 115,118, 119, or 121 of the invention comprises a N-hydroxy succinimide(NHS) ester linkage, oxime linkage, disulfide linkage, phosphoramidate,phosphorothioate, phosphorodithioate, phosphodiester linkage, or NHC(O),CH₃NC(O), CONH, C(O)NCH₃, S, SO, SO₂, O, NH, NCH₃ group. In anotherembodiment, the degradable linker, W and/or Y, of compounds havingFormulae Formulae 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100,101, 107, 108, 111, 114, 115, 118, 119, or 121 of the inventioncomprises a linker that is susceptible to cleavage by carboxypeptidaseactivity.

In another embodiment, W and/or Y of Formulae Formulae 43, 44, 46-52,58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115,118, 119, or 121 comprises a polyethylene glycol linker having Formula45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100.

In one embodiment, the nucleic acid conjugates of the instant inventionare assembled by solid phase synthesis, for example on an automatedpeptide synthesizer, for example a Miligen 9050 synthesizer and/or anautomated oligonucleotide synthesizer such as an ABI 394, 390Z, orPharmacia OligoProcess, OligoPilot, OligoMax, or AKTA synthesizer. Inanother embodiment, the nucleic acid conjugates of the invention areassembled post synthetically, for example, following solid phaseoligonucleotide synthesis (see for example FIG. 15).

In another embodiment, V of compounds having Formula 58-63 and 96comprise peptides having SEQ ID NOS: 14-23 (Table III).

In one embodiment, the nucleic acid conjugates of the instant inventionare assembled post synthetically, for example, following solid phaseoligonucleotide synthesis.

The present invention provides compositions and conjugates comprisingnucleosidic and non-nucleosidic derivatives. The present invention alsoprovides nucleic acid, polynucleotide and oligonucleotide derivativesincluding RNA, DNA, and PNA based conjugates. The attachment ofcompounds of the invention to nucleosides, nucleotides, non-nucleosides,and nucleic acid molecules is provided at any position within themolecule, for example, at internucleotide linkages, nucleosidic sugarhydroxyl groups such as 5′, 3′, and 2′-hydroxyls, and/or at nucleobasepositions such as amino and carbonyl groups.

The exemplary conjugates of the invention are described as compounds ofthe formulae herein, however, other peptide, protein, phospholipid, andpoly-alkyl glycol derivatives are provided by the invention, includingvarious analogs of the compounds of formulae 1-122, including but notlimited to different isomers of the compounds described herein.

In one embodiment, the present invention features molecules,compositions and conjugates of molecules, for example, non-nucleosidicsmall molecules, nucleosides, nucleotides, and nucleic acids, such asenzymatic nucleic acid molecules, antisense nucleic acids, 2-5Aantisense chimeras, triplex oligonucleotides, decoys, siNA, allozymes,aptamers, and antisense nucleic acids containing RNA cleaving chemicalgroups.

The exemplary folate conjugates of the invention are described ascompounds shown by formulae herein, however, other folate and antifolatederivatives are provided by the invention, including various folateanalogs of the formulae of the invention, including dihydrofloates,tetrahydrofolates, tetrahydrorpterins, folinic acid, pteropolyglutamicacid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacids. As used herein, the term “folate” is meant to refer to folate andfolate derivatives, including pteroic acid derivatives and analogs.

The present invention features compositions and conjugates to facilitatedelivery of molecules into a biological system such as cells. Theconjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosmall molecules, lipids, nucleosides, nucleotides, nucleic acids,negatively charged polymers and other polymers, for example proteins,peptides, carbohydrates, or polyamines. In general, the transportersdescribed are designed to be used either individually or as part of amulti-component system. The compounds of the invention generally shownin Formulae herein are expected to improve delivery of molecules into anumber of cell types originating from different tissues, in the presenceor absence of serum.

In another embodiment, the present invention features methods tomodulate gene expression, for example, genes involved in the progressionand/or maintenance of cancer or in a viral infection. For example, inone embodiment, the invention features the use of one or more of thenucleic acid-based molecules and methods independently or in combinationto inhibit the expression of the gene(s) encoding proteins associatedwith cancerous conditions, for example breast cancer, lung cancer,colorectal cancer, brain cancer, esophageal cancer, stomach cancer,bladder cancer, pancreatic cancer, cervical cancer, head and neckcancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrugresistant cancer associated genes.

In another embodiment, the invention features the use of one or more ofthe nucleic acid-based molecules and methods independently or incombination to inhibit the expression of the gene(s) encoding viralproteins, for example HIV, HBV, HCV, CMV, RSV, HSV, poliovirus,influenza, rhinovirus, west nile virus, Ebola virus, foot and mouthvirus, and papilloma virus associated genes.

In one embodiment, the invention features the use of an enzymaticnucleic acid molecule conjugate comprising compounds of formulae 1-122,preferably in the hammerhead, NCH, G-cleaver, amberzyme, zinzyme and/orDNAzyme motif, to inhibit the expression of cancer and virus associatedgenes.

In another embodiment, the invention features the use of an enzymaticnucleic acid molecule as a conjugate. These enzymatic nucleic acids cancatalyze the hydrolysis of RNA phosphodiester bonds in trans (and thuscan cleave other RNA molecules) under physiological conditions. Table Isummarizes some of the characteristics of these enzymatic nucleic acids.Without being bound by any particular theory, in general, enzymaticnucleic acids act by first binding to a target RNA. Such binding occursthrough the target binding portion of an enzymatic nucleic acid which isheld in close proximity to an enzymatic portion of the molecule thatacts to cleave the target RNA. Thus, the enzymatic nucleic acid firstrecognizes and then binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocut the target RNA. Strategic cleavage of such a target RNA destroys itsability to direct synthesis of an encoded protein. After an enzymaticnucleic acid has bound and cleaved its RNA target, it is released fromthat RNA to search for another target and can repeatedly bind and cleavenew targets. Thus, a single enzymatic nucleic acid molecule is able tocleave many molecules of target RNA. In addition, the enzymatic nucleicacid is a highly specific inhibitor of gene expression, with thespecificity of inhibition depending not only on the base-pairingmechanism of binding to the target RNA, but also on the mechanism oftarget RNA cleavage. Single mismatches, or base-substitutions, near thesite of cleavage can completely eliminate catalytic activity of anenzymatic nucleic acid.

In one embodiment of the invention described herein, the enzymaticnucleic acid molecule component of the conjugate is formed in ahammerhead or hairpin motif, but can also be formed in the motif of ahepatitis delta virus, group I intron, group II intron or RNase P RNA(in association with an RNA guide sequence), Neurospora VS RNA,DNAzymes, NCH cleaving motifs, or G-cleavers. Examples of suchhammerhead motifs are described by Dreyfus, supra, Rossi et al., 1992,AIDS Research and Human Retroviruses 8, 183; of hairpin motifs by Hampelet al., EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929,Feldstein et al., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene,82, 43, and Hampel et al., 1990 Nucleic Acids Res. 18, 299; Chowrira &McSwiggen, U.S. Pat. No. 5,631,359; of the hepatitis delta virus motifis described by Perrotta and Been, 1992 Biochemistry 31, 16; of theRNase P motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster andAltman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic Acids Res.24, 835; Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363);Group II introns are described by Griffin et al., 1995, Chem. Biol. 2,761; Michels and Pyle, 1995, Biochemistry 34, 2965; Pyle et al.,International PCT Publication No. WO 96/22689; of the Group I intron byCech et al., U.S. Pat. No. 4,987,071 and of DNAzymes by Usman et al.,International PCT Publication No. WO 95/11304; Chartrand et al., 1995,NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655; Santoro et al.,1997, PNAS 94, 4262, and Beigelman et al., International PCT publicationNo. WO 99/55857. NCH cleaving motifs are described in Ludwig & Sproat,International PCT Publication No. WO 98/58058; and G-cleavers aredescribed in Kore et al., 1998, Nucleic Acids Research 26, 4116-4120 andEckstein et al., International PCT Publication No. WO 99/16871.Additional motifs such as the Aptazyme (Breaker et al., WO 98/43993),Amberzyme (Class I motif; FIG. 3; Beigelman et al., U.S. Ser. No.09/301,511) and Zinzyme (FIG. 4) (Beigelman et al., U.S. Ser. No.09/301,511), all incorporated by reference herein including drawings,can also be used in the present invention. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule (Cech et al.,U.S. Pat. No. 4,987,071).

In one embodiment of the present invention, a nucleic acid moleculecomponent of a conjugate of the instant invention can be about 12 toabout 100 nucleotides in length. For example, enzymatic nucleic acidmolecules of the invention are preferably about 15 to about 50nucleotides in length, more preferably about 25 to about 40 nucleotidesin length, e.g., 34, 36, or 38 nucleotides in length (for example seeJarvis et al., 1996, J. Biol. Chem., 271, 29107-29112). ExemplaryDNAzymes of the invention are preferably about 15 to about 40nucleotides in length, more preferably about 25 to about 35 nucleotidesin length, e.g., 29, 30, 31, or 32 nucleotides in length (see forexample Santoro et al., 1998, Biochemistry, 37, 13330-13342; Chartrandet al., 1995, Nucleic Acids Research, 23, 4092-4096). Exemplaryantisense molecules of the invention are preferably about 15 to about 75nucleotides in length, more preferably about 20 to about 35 nucleotidesin length, e.g., 25, 26, 27, or 28 nucleotides in length (see, forexample, Woolf et al., 1992, PNAS., 89, 7305-7309; Milner et al., 1997,Nature Biotechnology, 15, 537-541). Exemplary triplex formingoligonucleotide molecules of the invention are preferably about 10 toabout 40 nucleotides in length, more preferably about 12 to about 25nucleotides in length, e.g., 18, 19, 20, or 21 nucleotides in length(see for example Maher et al., 1990, Biochemistry, 29, 8820-8826;Strobel and Dervan, 1990, Science, 249, 73-75). Exemplary doublestranded siNA molecules of the invention comprise about 19 to about 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, 24, or 25nucleotides in length, for each strand of the siNA molecule. Exemplarysingle stranded siNA molecules of the invention are about 38 to about 50nucleotides in length, e.g., about 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 nucleotides in length. The length of the nucleic acidmolecules described and exemplified herein are not limiting within thegeneral size ranges stated.

The conjugates of the invention are added directly, or can be complexedwith cationic lipids, packaged within liposomes, or otherwise deliveredto target cells or tissues. The conjugates and/or conjugate complexescan be locally administered to relevant tissues ex vivo, or in vivothrough injection or infusion pump, with or without their incorporationin biopolymers. The compositions and conjugates of the instantinvention, individually, or in combination or in conjunction with otherdrugs, can be used to treat diseases or conditions discussed above. Forexample, to treat a disease or condition associated with the levels of apathogenic protein, the patient can be treated, or other appropriatecells can be treated, as is evident to those skilled in the art,individually or in combination with one or more drugs under conditionssuitable for the treatment.

In a further embodiment, the described molecules can be used incombination with other known treatments to treat conditions or diseasesdiscussed above. For example, the described molecules can be used incombination with one or more known therapeutic agents to treat breast,lung, prostate, colorectal, brain, esophageal, bladder, pancreatic,cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma,multidrug resistant cancers, and/or HIV, HBV, HCV, CMV, RSV, HSV,poliovirus, influenza, rhinovirus, west nile virus, Ebola virus, footand mouth virus, and papilloma virus infection.

Included in another embodiment are a series of multi-domain cellulartransport vehicles (MCTV) including one or more compounds of Formulae1-122 herein that enhance the cellular uptake and transmembranepermeability of negatively charged molecules in a variety of cell types.The compounds of the invention are used either alone or in combinationwith other compounds with a neutral or a negative charge including butnot limited to neutral lipid and/or targeting components, to improve theeffectiveness of the formulation or conjugate in delivering andtargeting the predetermined compound or molecule to cells. Anotherembodiment of the invention encompasses the utility of these compoundsfor increasing the transport of other impermeable and/or lipophiliccompounds into cells. Targeting components include ligands for cellsurface receptors including, peptides and proteins, glycolipids, lipids,carbohydrates, and their synthetic variants, for example folatereceptors.

In another embodiment, the compounds of the invention are provided as asurface component of a lipid aggregate, such as a liposome encapsulatedwith the predetermined molecule to be delivered. Liposomes, which can beunilamellar or multilamellar, can introduce encapsulated material into acell by different mechanisms. For example, the liposome can directlyintroduce its encapsulated material into the cell cytoplasm by fusingwith the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) and itscontents released from the liposome and out of the acidic vacuole intothe cellular cytoplasm.

In one embodiment the invention features a lipid aggregate formulationof the compounds described herein, including phosphatidylcholine (ofvarying chain length; e.g., egg yolk phosphatidylcholine), cholesterol,a cationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000(DSPE-PEG2000). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In another embodimentthis cationic lipid aggregate comprises a covalently bound compounddescribed in any of the Formulae herein.

In another embodiment, polyethylene glycol (PEG) is covalently attachedto the compounds of the present invention. The attached PEG can be anymolecular weight but is preferably between 2000-50,000 daltons.

The compounds and methods of the present invention are useful forintroducing nucleotides, nucleosides, nucleic acid molecules, lipids,peptides, proteins, and/or non-nucleosidic small molecules into a cell.For example, the invention can be used for nucleotide, nucleoside,nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic smallmolecule delivery where the corresponding target site of action existsintracellularly.

In one embodiment, the compounds of the instant invention provideconjugates of molecules that can interact with cellular receptors, suchas high affinity folate receptors and ASGPr receptors, and provide anumber of features that allow the efficient delivery and subsequentrelease of conjugated compounds across biological membranes. Thecompounds utilize chemical linkages between the receptor ligand and thecompound to be delivered of length that can interact preferentially withcellular receptors. Furthermore, the chemical linkages between theligand and the compound to be delivered can be designed as degradablelinkages, for example by utilizing a phosphate linkage that is proximalto a nucleophile, such as a hydroxyl group. Deprotonation of thehydroxyl group or an equivalent group, as a result of pH or interactionwith a nuclease, can result in nucleophilic attack of the phosphateresulting in a cyclic phosphate intermediate that can be hydrolyzed.This cleavage mechanism is analogous RNA cleavage in the presence of abase or RNA nuclease. Alternately, other degradable linkages can beselected that respond to various factors such as UV irradiation,cellular nucleases, pH, temperature etc. The use of degradable linkagesallows the delivered compound to be released in a predetermined system,for example in the cytoplasm of a cell, or in a particular cellularorganelle.

The present invention also provides ligand derived phosphoramidites thatare readily conjugated to compounds and molecules of interest.Phosphoramidite compounds of the invention permit the direct attachmentof conjugates to molecules of interest without the need for usingnucleic acid phosphoramidite species as scaffolds. As such, the used ofphosphoramidite chemistry can be used directly in coupling the compoundsof the invention to a compound of interest, without the need for othercondensation reactions, such as condensation of the ligand to an aminogroup on the nucleic acid, for example at the N6 position of adenosineor a 2′-deoxy-2′-amino function. Additionally, compounds of theinvention can be used to introduce non-nucleic acid based conjugatedlinkages into oligonucleotides that can provide more efficient couplingduring oligonucleotide synthesis than the use of nucleic acid-basedphosphoramidites. This improved coupling can take into account improvedsteric considerations of abasic or non-nucleosidic scaffolds bearingpendant alkyl linkages.

Compounds of the invention utilizing triphosphate groups can be utilizedin the enzymatic incorporation of conjugate molecules intooligonucleotides. Such enzymatic incorporation is useful when conjugatesare used in post-synthetic enzymatic conjugation or selection reactions,(see for example Matulic-Adamic et al., 2000, Bioorg. Med. Chem. Lett.,10, 1299-1302; Lee et al., 2001, NAR., 29, 1565-1573; Joyce, 1989, Gene,82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992,Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268;Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17,89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra;Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish etal., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr. Opin.Chem. Biol., 4, 669).

Compounds of the invention can be used to detect the presence of atarget molecule in a biological system, such as tissue, cell or celllysate. Examples of target molecules include nucleic acids, proteins,peptides, antibodies, polysaccharides, lipids, hormones, sugars, metals,microbial or cellular metabolites, analytes, pharmaceuticals, and otherorganic and inorganic molecules or other biomolecules in a sample. Thecompounds of the instant invention can be conjugated to a predeterminedcompound or molecule that is capable of interacting with the targetmolecule in the system and providing a detectable signal or response.Various compounds and molecules known in the art that can be used inthese applications include but are not limited to antibodies, labeledantibodies, allozymes, aptamers, labeled nucleic acid probes, molecularbeacons, fluorescent molecules, radioisotopes, polysaccharides, and anyother compound capable of interacting with the target molecule andgenerating a detectable signal upon target interaction. For example,such compounds are described in Application entitled “NUCLEIC ACIDSENSOR MOLECULES”, U.S. Ser. No. 09/800,594 filed on Mar. 6, 2001 withinventors Nassim Usman and James A. McSwiggen, which is incorporated byreference in its entirety, including the drawings.

The term “biodegradable nucleic acid linker molecule” as used herein,refers to a nucleic acid molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule. The stability of the biodegradable nucleicacid linker molecule can be modulated by using various combinations ofribonucleotides, deoxyribonucleotides, and chemically modifiednucleotides, for example 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino,2′-O-allyl, 2′-O-allyl, and other 2′-modified or base modifiednucleotides. The biodegradable nucleic acid linker molecule can be adimer, trimer, tetramer or longer nucleic acid molecule, for example anoligonucleotide of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 nucleotides in length, or can comprise a singlenucleotide with a phosphorus based linkage, for example aphosphoramidate or phosphodiester linkage. The biodegradable nucleicacid linker molecule can also comprise nucleic acid backbone, nucleicacid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive molecules contemplated by the instant invention includetherapeutically active molecules such as antibodies, hormones,antivirals, peptides, proteins, chemotherapeutics, small molecules,vitamins, co-factors, nucleosides, nucleotides, oligonucleotides,enzymatic nucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

The term “nitrogen containing group” as used herein refers to anychemical group or moiety comprising a nitrogen or substituted nitrogen.Non-limiting examples of nitrogen containing groups include amines,substituted amines, amides, alkylamines, amino acids such as arginine orlysine, polyamines such as spermine or spermidine, cyclic amines such aspyridines, pyrimidines including uracil, thymine, and cytosine,morpholines, phthalimides, and heterocyclic amines such as purines,including guanine and adenine.

The term “target molecule” as used herein, refers to nucleic acidmolecules, proteins, peptides, antibodies, polysaccharides, lipids,sugars, metals, microbial or cellular metabolites, analytes,pharmaceuticals, and other organic and inorganic molecules that arepresent in a system.

By “inhibit” or “down-regulate” it is meant that the expression of thegene, or level of RNAs or equivalent RNAs encoding one or more proteinsubunits, or activity of one or more protein subunits, such aspathogenic protein, viral protein or cancer related protein subunit(s),is reduced below that observed in the absence of the compounds orcombination of compounds of the invention. In one embodiment, inhibitionor down-regulation with an enzymatic nucleic acid molecule preferably isbelow that level observed in the presence of an enzymatically inactiveor attenuated molecule that is able to bind to the same site on thetarget RNA, but is unable to cleave that RNA. In another embodiment,inhibition or down-regulation with antisense oligonucleotides ispreferably below that level observed in the presence of, for example, anoligonucleotide with scrambled sequence or with mismatches. In anotherembodiment, inhibition or down-regulation of viral or oncogenic RNA,protein, or protein subunits with a compound of the instant invention isgreater in the presence of the compound than in its absence.

By “up-regulate” is meant that the expression of the gene, or level ofRNAs or equivalent RNAs encoding one or more protein subunits, oractivity of one or more protein subunits, such as viral or oncogenicprotein subunit(s), is greater than that observed in the absence of thecompounds or combination of compounds of the invention. For example, theexpression of a gene, such as a viral or cancer related gene, can beincreased in order to treat, prevent, ameliorate, or modulate apathological condition caused or exacerbated by an absence or low levelof gene expression.

By “modulate” is meant that the expression of the gene, or level of RNAsor equivalent RNAs encoding one or more protein subunits, or activity ofone or more protein subunit(s) of a protein, for example a viral orcancer related protein is up-regulated or down-regulated, such that theexpression, level, or activity is greater than or less than thatobserved in the absence of the compounds or combination of compounds ofthe invention.

The term “enzymatic nucleic acid molecule” as used herein refers to anucleic acid molecule which has complementarity in a substrate bindingregion to a specified gene target, and also has an enzymatic activitywhich is active to specifically cleave target RNA. That is, theenzymatic nucleic acid molecule is able to intermolecularly cleave RNAand thereby inactivate a target RNA molecule. These complementaryregions allow sufficient hybridization of the enzymatic nucleic acidmolecule to the target RNA and thus permit cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% canalso be useful in this invention (see for example Werner and Uhlenbeck,1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999,Antisense and Nucleic Acid Drug Dev., 9, 25-31). The nucleic acids canbe modified at the base, sugar, and/or phosphate groups. The termenzymatic nucleic acid is used interchangeably with phrases such asribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme oraptamer-binding ribozyme, regulatable ribozyme, catalyticoligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease,endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of theseterminologies describe nucleic acid molecules with enzymatic activity.The specific enzymatic nucleic acid molecules described in the instantapplication are not limiting in the invention and those skilled in theart will recognize that all that is important in an enzymatic nucleicacid molecule of this invention is that it has a specific substratebinding site which is complementary to one or more of the target nucleicacid regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart a nucleic acidcleaving and/or ligation activity to the molecule (Cech et al., U.S.Pat. No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).

The term “nucleic acid molecule” as used herein, refers to a moleculehaving nucleotides. The nucleic acid can be single, double, or multiplestranded and can comprise modified or unmodified nucleotides ornon-nucleotides or various mixtures and combinations thereof.

The term “enzymatic portion” or “catalytic domain” as used herein refersto that portion/region of the enzymatic nucleic acid molecule essentialfor cleavage of a nucleic acid substrate (for example see FIG. 1).

The term “substrate binding arm” or “substrate binding domain” as usedherein refers to that portion/region of a enzymatic nucleic acid whichis able to interact, for example via complementarity (i.e., able tobase-pair with), with a portion of its substrate. Preferably, suchcomplementarity is 100%, but can be less if desired. For example, as fewas 10 bases out of 14 can be base-paired (see for example Werner andUhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al.,1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). Examples of sucharms are shown generally in FIGS. 1-4. That is, these arms containsequences within a enzymatic nucleic acid which are intended to bringenzymatic nucleic acid and target RNA together through complementarybase-pairing interactions. The enzymatic nucleic acid of the inventioncan have binding arms that are contiguous or non-contiguous and can beof varying lengths. The length of the binding arm(s) are preferablygreater than or equal to four nucleotides and of sufficient length tostably interact with the target RNA; preferably 12-100 nucleotides; morepreferably 14-24 nucleotides long (see for example Werner and Uhlenbeck,supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herranceet al., 1993, EMBO J., 12, 2567-73). If two binding arms are chosen, thedesign is such that the length of the binding arms are symmetrical(i.e., each of the binding arms is of the same length; e.g., five andfive nucleotides, or six and six nucleotides, or seven and sevennucleotides long) or asymmetrical (i.e., the binding arms are ofdifferent length; e.g., six and three nucleotides; three and sixnucleotides long; four and five nucleotides long; four and sixnucleotides long; four and seven nucleotides long; and the like).

The term “Inozyme” or “NCH” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described asNCH Rz in FIG. 1. Inozymes possess endonuclease activity to cleave RNAsubstrates having a cleavage triplet NCH/, where N is a nucleotide, C iscytidine and H is adenosine, uridine or cytidine, and/represents thecleavage site. H is used interchangeably with X. Inozymes can alsopossess endonuclease activity to cleave RNA substrates having a cleavagetriplet NCN/, where N is a nucleotide, C is cytidine, and/represents thecleavage site. “I” in FIG. 2 represents an Inosine nucleotide,preferably a ribo-Inosine or xylo-Inosine nucleoside.

The term “G-cleaver” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described asG-cleaver Rz in FIG. 1. G-cleavers possess endonuclease activity tocleave RNA substrates having a cleavage triplet NYN/, where N is anucleotide, Y is uridine or cytidine and/represents the cleavage site.G-cleavers can be chemically modified as is generally shown in FIG. 2.

The term “amberzyme” motif as used herein, refers to an enzymaticnucleic acid molecule comprising a motif as is generally described inFIG. 2. Amberzymes possess endonuclease activity to cleave RNAsubstrates having a cleavage triplet NG/N, where N is a nucleotide, G isguanosine, and/represents the cleavage site. Amberzymes can bechemically modified to increase nuclease stability through substitutionsas are generally shown in FIG. 3. In addition, differing nucleosideand/or non-nucleoside linkers can be used to substitute the 5′-gaaa-3′loops shown in the figure. Amberzymes represent a non-limiting exampleof an enzymatic nucleic acid molecule that does not require aribonucleotide (2′-OH) group within its own nucleic acid sequence foractivity.

The term “zinzyme” motif as used herein, refers to an enzymatic nucleicacid molecule comprising a motif as is generally described in FIG. 3.Zinzymes possess endonuclease activity to cleave RNA substrates having acleavage triplet including but not limited to YG/Y, where Y is uridineor cytidine, and G is guanosine and/represents the cleavage site.Zinzymes can be chemically modified to increase nuclease stabilitythrough substitutions as are generally shown in FIG. 3, includingsubstituting 2′-O-methyl guanosine nucleotides for guanosinenucleotides. In addition, differing nucleotide and/or non-nucleotidelinkers can be used to substitute the 5′-gaaa-2′ loop shown in thefigure. Zinzymes represent a non-limiting example of an enzymaticnucleic acid molecule that does not require a ribonucleotide (2′-OH)group within its own nucleic acid sequence for activity.

The term ‘DNAzyme’ as used herein, refers to an enzymatic nucleic acidmolecule that does not require the presence of a 2′-OH group for itsactivity. In particular embodiments the enzymatic nucleic acid moleculecan have an attached linker(s) or other attached or associated groups,moieties, or chains containing one or more nucleotides with 2′-OHgroups. DNAzymes can be synthesized chemically or expressed endogenouslyin vivo, by means of a single stranded DNA vector or equivalent thereof.An example of a DNAzyme is shown in FIG. 4 and is generally reviewed inUsman et al., International PCT Publication No. WO 95/11304; Chartrandet al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio. 2, 655;Santoro et al., 1997, PNAS 94, 4262; Breaker, 1999, NatureBiotechnology, 17, 422-423; and Santoro et. al., 2000, J. Am. Chem.Soc., 122, 2433-39. Additional DNAzyme motifs can be selected for usingtechniques similar to those described in these references, and hence,are within the scope of the present invention.

The term “sufficient length” as used herein, refers to anoligonucleotide of length great enough to provide the intended functionunder the expected condition, i.e., greater than or equal to 3nucleotides. For example, for binding arms of enzymatic nucleic acid“sufficient length” means that the binding arm sequence is long enoughto provide stable binding to a target site under the expected bindingconditions. Preferably, the binding arms are not so long as to preventuseful turnover of the nucleic acid molecule.

The term “stably interact” as used herein, refers to interaction of theoligonucleotides with target nucleic acid (e.g., by forming hydrogenbonds with complementary nucleotides in the target under physiologicalconditions) that is sufficient to the intended purpose (e.g., cleavageof target RNA by an enzyme).

The term “homology” as used herein, refers to the nucleotide sequence oftwo or more nucleic acid molecules is partially or completely identical.

The term “antisense nucleic acid”, as used herein, refers to anon-enzymatic nucleic acid molecule that binds to target RNA by means ofRNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993Nature 365, 566) interactions and alters the activity of the target RNA(for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf etal., U.S. Pat. No. 5,849,902). Typically, antisense molecules arecomplementary to a target sequence along a single contiguous sequence ofthe antisense molecule. However, in certain embodiments, an antisensemolecule can bind to substrate such that the substrate molecule forms aloop, and/or an antisense molecule can bind such that the antisensemolecule forms a loop. Thus, the antisense molecule can be complementaryto two (or even more) non-contiguous substrate sequences or two (or evenmore) non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. For a review of currentantisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274,21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al.,1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol.,313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157, Crooke,1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA can be usedto target RNA by means of DNA-RNA interactions, thereby activating RNaseH, which digests the target RNA in the duplex. The antisenseoligonucleotides can comprise one or more RNAse H activating region,which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof.

The term “RNase H activating region” as used herein, refers to a region(generally greater than or equal to 4-25 nucleotides in length,preferably from 5-11 nucleotides in length) of a nucleic acid moleculecapable of binding to a target RNA to form a non-covalent complex thatis recognized by cellular RNase H enzyme (see for example Arrow et al.,U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). TheRNase H enzyme binds to the nucleic acid molecule-target RNA complex andcleaves the target RNA sequence. The RNase H activating regioncomprises, for example, phosphodiester, phosphorothioate (preferably atleast four of the nucleotides are phosphorothiote substitutions; morespecifically, 4-11 of the nucleotides are phosphorothiotesubstitutions); phosphorodithioate, 5′-thiophosphate, ormethylphosphonate backbone chemistry or a combination thereof. Inaddition to one or more backbone chemistries described above, the RNaseH activating region can also comprise a variety of sugar chemistries.For example, the RNase H activating region can comprise deoxyribose,arabino, fluoroarabino or a combination thereof, nucleotide sugarchemistry. Those skilled in the art will recognize that the foregoingare non-limiting examples and that any combination of phosphate, sugarand base chemistry of a nucleic acid that supports the activity of RNaseH enzyme is within the scope of the definition of the RNase H activatingregion and the instant invention.

The term “2-5A antisense chimera” as used herein, refers to an antisenseoligonucleotide containing a 5′-phosphorylated 2′-5′-linked adenylateresidue. These chimeras bind to target RNA in a sequence-specific mannerand activate a cellular 2-5A-dependent ribonuclease which, in turn,cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Playerand Torrence, 1998, Pharmacol. Ther., 78, 55-113).

The term “triplex forming oligonucleotides” as used herein, refers to anoligonucleotide that can bind to a double-stranded DNA in asequence-specific manner to form a triple-strand helix. Formation ofsuch triple helix structure has been shown to inhibit transcription ofthe targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci.USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al.,2000, Biochim. Biophys. Acta, 1489, 181-206).

The term “gene” it as used herein, refers to a nucleic acid that encodesan RNA, for example, nucleic acid sequences including but not limited tostructural genes encoding a polypeptide.

The term “pathogenic protein” as used herein, refers to endogenous orexongenous proteins that are associated with a disease state orcondition, for example a particular cancer or viral infection.

The term “complementarity” refers to the ability of a nucleic acid toform hydrogen bond(s) with another RNA sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., enzymatic nucleic acid cleavage, antisense or triplehelix inhibition. Determination of binding free energies for nucleicacid molecules is well known in the art (see, e.g., Turner et al., 1987,CSH Symp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat.Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule which can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%,90%, and 100% complementary). “Perfectly complementary” means that allthe contiguous residues of a nucleic acid sequence will hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence.

The term “RNA” as used herein, refers to a molecule comprising at leastone ribonucleotide residue. By “ribonucleotide” or “2′-OH” is meant anucleotide with a hydroxyl group at the 2′ position of aβ-D-ribo-furanose moiety.

The term “decoy RNA” as used herein, refers to a RNA molecule or aptamerthat is designed to preferentially bind to a predetermined ligand. Suchbinding can result in the inhibition or activation of a target molecule.The decoy RNA or aptamer can compete with a naturally occurring bindingtarget for the binding of a specific ligand. For example, it has beenshown that over-expression of HIV trans-activation response (TAR) RNAcan act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA(Sullenger et al., 1990, Cell, 63, 601-608). This is but a specificexample and those in the art will recognize that other embodiments canbe readily generated using techniques generally known in the art, seefor example Gold et al., 1995, Annu. Rev. Biochem., 64, 763; Brody andGold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2,100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000,Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.Similarly, a decoy RNA can be designed to bind to a receptor and blockthe binding of an effector molecule or a decoy RNA can be designed tobind to receptor of interest and prevent interaction with the receptor.

The term “single stranded RNA” (ssRNA) as used herein refers to anaturally occurring or synthetic ribonucleic acid molecule comprising alinear single strand, for example a ssRNA can be a messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA) etc. of a gene.

The term “single stranded DNA” (ssDNA) as used herein refers to anaturally occurring or synthetic deoxyribonucleic acid moleculecomprising a linear single strand, for example, a ssDNA can be a senseor antisense gene sequence or EST (Expressed Sequence Tag).

The term “double stranded RNA” or “dsRNA” as used herein refers to adouble stranded RNA molecule capable of RNA interference, includingshort interfering RNA (siNA).

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Bass,2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;and Kreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Nonlimiting examples of siNA molecules of the invention are described inHaeberli et al., PCT/US03/05346 and McSwiggen et al., PCT/US03/05028,both incorporated by reference herein in their entirety including thedrawings, and in FIGS. 34-42 herein. Chemical modifications described inHaeberli et al., PCT/US03/05346 and McSwiggen et al., PCT/US03/05028and/or shown in Table IV can be applied to any siNA sequence of theinvention. For example the siNA can be a double-stranded polynucleotidemolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary (i.e.each strand comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 19 base pairs); theantisense strand comprises nucleotide sequence that is complementary tonucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense strand comprises nucleotide sequence correspondingto the target nucleic acid sequence or a portion thereof. Alternatively,the siNA is assembled from a single oligonucleotide, where theself-complementary sense and antisense regions of the siNA are linked bymeans of a nucleic acid based or non-nucleic acid-based linker(s). ThesiNA can be a polynucleotide with a hairpin secondary structure, havingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a separate target nucleic acid molecule or a portion thereofand the sense region having nucleotide sequence corresponding to thetarget nucleic acid sequence or a portion thereof. The siNA can be acircular single-stranded polynucleotide having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises nucleotide sequence thatis complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active siNA molecule capable ofmediating RNAi. The siNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (forexample, where such siNA molecule does not require the presence withinthe siNA molecule of nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell., 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or5′,3′-diphosphate. In certain embodiment, the siNA molecule of theinvention comprises separate sense and antisense sequences or regions,wherein the sense and antisense regions are covalently linked bynucleotide or non-nucleotide linkers molecules as is known in the art,or are alternately non-covalently linked by ionic interactions, hydrogenbonding, van der waals interactions, hydrophobic interactions, and/orstacking interactions. In certain embodiments, the siNA molecules of theinvention comprise nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, the siNAmolecule of the invention interacts with nucleotide sequence of a targetgene in a manner that causes inhibition of expression of the targetgene. As used herein, siNA molecules need not be limited to thosemolecules containing only RNA, but further encompasseschemically-modified nucleotides and non-nucleotides. In certainembodiments, the short interfering nucleic acid molecules of theinvention lack 2′-hydroxy (2′-OH) containing nucleotides. Applicantdescribes in certain embodiments short interfering nucleic acids that donot require the presence of nucleotides having a 2′-hydroxy group formediating RNAi and as such, short interfering nucleic acid molecules ofthe invention optionally do not include any ribonucleotides (e.g.,nucleotides having a 2′-OH group). Such siNA molecules that do notrequire the presence of ribonucleotides within the siNA molecule tosupport RNAi can however have an attached linker or linkers or otherattached or associated groups, moieties, or chains containing one ormore nucleotides with 2′-OH groups. Optionally, siNA molecules cancomprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of thenucleotide positions. The modified short interfering nucleic acidmolecules of the invention can also be referred to as short interferingmodified oligonucleotides “siMON.” As used herein, the term siNA ismeant to be equivalent to other terms used to describe nucleic acidmolecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically-modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), translationalsilencing, and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing, orepigenetics. For example, siNA molecules of the invention can be used toepigenetically silence genes at both the post-transcriptional level orthe pre-transcriptional level. In a non-limiting example, epigeneticregulation of gene expression by siNA molecules of the invention canresult from siNA mediated modification of chromatin structure to altergene expression (see, for example, Allshire, 2002, Science, 297,1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002,Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

The term “allozyme” as used herein refers to an allosteric enzymaticnucleic acid molecule, see for example see for example George et al.,U.S. Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No.5,589,332, Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

The term “cell” as used herein, refers to its usual biological sense,and does not refer to an entire multicellular organism. The cell can,for example, be in vitro, e.g., in cell culture, or present in amulticellular organism, including, e.g., birds, plants and mammals suchas humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cellcan be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalianor plant cell).

The term “highly conserved sequence region” as used herein, refers to anucleotide sequence of one or more regions in a target gene does notvary significantly from one generation to the other or from onebiological system to the other.

The term “non-nucleotide” as used herein, refers to any group orcompound which can be incorporated into a nucleic acid chain in theplace of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound is abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine.

The term “nucleotide” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are recognized in the art to include natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of chemically modified and other natural nucleicacid bases that can be introduced into nucleic acids include, forexample, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

The term “nucleoside” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a sugar. Nucleosides arerecognized in the art to include natural bases (standard), and modifiedbases well known in the art. Such bases are generally located at the 1′position of a nucleoside sugar moiety. Nucleosides generally comprise abase and sugar group. The nucleosides can be unmodified or modified atthe sugar, and/or base moiety, (also referred to interchangeably asnucleoside analogs, modified nucleosides, non-natural nucleosides,non-standard nucleosides and other; see for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra all are hereby incorporated by reference herein).There are several examples of modified nucleic acid bases known in theart as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.Some of the non-limiting examples of chemically modified and othernatural nucleic acid bases that can be introduced into nucleic acidsinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguano sine, N6-methyladenosine, 7-methylguano sine, 5-methoxyaminomethyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyluridine,5-methyloxyuridine, 5-methyl-2-thiouridine,2-methylthio-N-6-isopentenyladenosine, beta-D-mannosylqueosine,uridine-5-oxyacetic acid, 2-thiocytidine, threonine derivatives andothers (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman,supra). By “modified bases” in this aspect is meant nucleoside basesother than adenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases can be used at any position, for example, withinthe catalytic core of an enzymatic nucleic acid molecule and/or in thesubstrate-binding regions of the nucleic acid molecule.

The term “cap structure” as used herein, refers to chemicalmodifications, which have been incorporated at either terminus of theoligonucleotide (see for example Wincott et al., WO 97/26270,incorporated by reference herein). These terminal modifications protectthe nucleic acid molecule from exonuclease degradation, and can help indelivery and/or localization within a cell. The cap can be present atthe 5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can bepresent on both terminus. In non-limiting examples, the 5′-cap includesinverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

The term “abasic” as used herein, refers to sugar moieties lacking abase or having other chemical groups in place of a base at the 1′position, for example a 3′,3′-linked or 5′,5′-linked deoxyabasic ribosederivative (for more details see Wincott et al., International PCTpublication No. WO 97/26270).

The term “unmodified nucleoside” as used herein, refers to one of thebases adenine, cytosine, guanine, thymine, uracil joined to the 1′carbon of β-D-ribo-furanose.

The term “modified nucleoside” as used herein, refers to any nucleotidebase which contains a modification in the chemical structure of anunmodified nucleotide base, sugar and/or phosphate.

The term “consists essentially of” as used herein, is meant that theactive nucleic acid molecule of the invention, for example, an enzymaticnucleic acid molecule, contains an enzymatic center or core equivalentto those in the examples, and binding arms able to bind RNA such thatcleavage at the target site occurs. Other sequences can be present whichdo not interfere with such cleavage. Thus, a core region can, forexample, include one or more loop, stem-loop structure, or linker whichdoes not prevent enzymatic activity. For example, a core sequence for ahammerhead enzymatic nucleic acid can comprise a conserved sequence,such as 5′-CUGAUGAG-3′ and 5′-CGAA-3′ connected by “X”, where X is5′-GCCGUUAGGC-3′ (SEQ ID NO 1), or any other Stem II region known in theart, or a nucleotide and/or non-nucleotide linker. Similarly, for othernucleic acid molecules of the instant invention, such as Inozyme,G-cleaver, amberzyme, zinzyme, DNAzyme, antisense, 2-5A antisense,triplex forming nucleic acid, and decoy nucleic acids, other sequencesor non-nucleotide linkers can be present that do not interfere with thefunction of the nucleic acid molecule.

Sequence X can be a linker of >2 nucleotides in length, preferably 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 26, 30, where the nucleotides can preferablybe internally base-paired to form a stem of preferably >2 base pairs. Inyet another embodiment, the nucleotide linker X can be a nucleic acidaptamer, such as an ATP aptamer, HIV Rev aptamer (RRE), HIV Tat aptamer(TAR) and others (for a review see Gold et al., 1995, Annu. Rev.Biochem., 64, 763; and Szostak & Ellington, 1993, in The RNA World, ed.Gesteland and Atkins, pp. 511, CSH Laboratory Press). A “nucleic acidaptamer” as used herein is meant to indicate a nucleic acid sequencecapable of interacting with a ligand. The ligand can be any natural or asynthetic molecule, including but not limited to a resin, metabolites,nucleosides, nucleotides, drugs, toxins, transition state analogs,peptides, lipids, proteins, amino acids, nucleic acid molecules,hormones, carbohydrates, receptors, cells, viruses, bacteria and others.

Alternatively or in addition, sequence X can be a non-nucleotide linker.Non-nucleotides can include abasic nucleotide, polyether, polyamine,polyamide, peptide, carbohydrate, lipid, or polyhydrocarbon compounds.Specific examples include those described by Seela and Kaiser, NucleicAcids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload andSchepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz,J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic AcidsRes. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.,Biochemistry 1991, 30:9914; Arnold et al., International Publication No.WO 89/02439; Usman et al., International Publication No. WO 95/06731;Dudycz et al., International Publication No. WO 95/11910 and Ferentz andVerdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated byreference herein. A “non-nucleotide” further means any group or compoundwhich can be incorporated into a nucleic acid chain in the place of oneor more nucleotide units, including either sugar and/or phosphatesubstitutions, and allows the remaining bases to exhibit their enzymaticactivity. The group or compound can be abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine. Thus, in a preferred embodiment,the invention features an enzymatic nucleic acid molecule having one ormore non-nucleotide moieties, and having enzymatic activity to cleave anRNA or DNA molecule.

The term “patient” as used herein, refers to an organism, which is adonor or recipient of explanted cells or the cells themselves. “Patient”also refers to an organism to which the nucleic acid molecules of theinvention can be administered. Preferably, a patient is a mammal ormammalian cells. More preferably, a patient is a human or human cells.

The term “enhanced enzymatic activity” as used herein, includes activitymeasured in cells and/or in vivo where the activity is a reflection ofboth the catalytic activity and the stability of the nucleic acidmolecules of the invention. In this invention, the product of theseproperties can be increased in vivo compared to an all RNA enzymaticnucleic acid or all DNA enzyme. In some cases, the activity or stabilityof the nucleic acid molecule can be decreased (i.e., less thanten-fold), but the overall activity of the nucleic acid molecule isenhanced, in vivo.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and can or can not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements can be present.

The term “negatively charged molecules” as used herein, refers tomolecules such as nucleic acid molecules (e.g., RNA, DNA,oligonucleotides, mixed polymers, peptide nucleic acid, and the like),peptides (e.g., polyaminoacids, polypeptides, proteins and the like),nucleotides, pharmaceutical and biological compositions, that havenegatively charged groups that can ion-pair with the positively chargedhead group of the cationic lipids of the invention.

The term “coupling” as used herein, refers to a reaction, eitherchemical or enzymatic, in which one atom, moiety, group, compound ormolecule is joined to another atom, moiety, group, compound or molecule.

The terms “deprotection” or “deprotecting” as used herein, refers to theremoval of a protecting group.

The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain “isoalkyl”, andcyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio,alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom about 1 to about 7 carbons, more preferably about 1 to about 4carbons. The alkyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” alsoincludes alkenyl groups containing at least one carbon-carbon doublebond, including straight-chain, branched-chain, and cyclic groups.Preferably, the alkenyl group has about 2 to about 12 carbons. Morepreferably it is a lower alkenyl of from about 2 to about 7 carbons,more preferably about 2 to about 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl,alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substitutedaryl groups. The term “alkyl” also includes alkynyl groups containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group hasabout 2 to about 12 carbons. More preferably it is a lower alkynyl offrom about 2 to about 7 carbons, more preferably about 2 to about 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moietiesof the invention can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. The preferred substituent(s)of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano,alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” grouprefers to an alkyl group (as described above) covalently joined to anaryl group (as described above). Carbocyclic aryl groups are groupswherein the ring atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted. Heterocyclic aryl groups aregroups having from about 1 to about 3 heteroatoms as ring atoms in thearomatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, methoxyethyl or ethoxymethyl.

The term “alkyl-thio-alkyl” as used herein refers to an alkyl-5-alkylthioether, for example, methylthiomethyl or methylthioethyl.

The term “amino” as used herein refers to a nitrogen containing group asis known in the art derived from ammonia by the replacement of one ormore hydrogen radicals by organic radicals. For example, the terms“aminoacyl” and “aminoalkyl” refer to specific N-substituted organicradicals with acyl and alkyl substituent groups respectively.

The term “amination” as used herein refers to a process in which anamino group or substituted amine is introduced into an organic molecule.

The term “exocyclic amine protecting moiety” as used herein refers to anucleobase amino protecting group compatible with oligonucleotidesynthesis, for example, an acyl or amide group.

The term “alkenyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl,and 2-methyl-3-heptene.

The term “alkoxy” as used herein refers to an alkyl group of indicatednumber of carbon atoms attached to the parent molecular moiety throughan oxygen bridge. Examples of alkoxy groups include, for example,methoxy, ethoxy, propoxy and isopropoxy.

The term “alkynyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon triple bond. Examples of “alkynyl” include propargyl,propyne, and 3-hexyne.

The term “aryl” as used herein refers to an aromatic hydrocarbon ringsystem containing at least one aromatic ring. The aromatic ring canoptionally be fused or otherwise attached to other aromatic hydrocarbonrings or non-aromatic hydrocarbon rings. Examples of aryl groupsinclude, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthaleneand biphenyl. Preferred examples of aryl groups include phenyl andnaphthyl.

The term “cycloalkenyl” as used herein refers to a C3-C8 cyclichydrocarbon containing at least one carbon-carbon double bond. Examplesof cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “cycloalkyl” as used herein refers to a C3-C8 cyclichydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “cycloalkylalkyl,” as used herein, refers to a C3-C7 cycloalkylgroup attached to the parent molecular moiety through an alkyl group, asdefined above. Examples of cycloalkylalkyl groups includecyclopropylmethyl and cyclopentylethyl.

The terms “halogen” or “halo” as used herein refers to indicatefluorine, chlorine, bromine, and iodine.

The term “heterocycloalkyl,” as used herein refers to a non-aromaticring system containing at least one heteroatom selected from nitrogen,oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused toor otherwise attached to other heterocycloalkyl rings and/ornon-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups havefrom 3 to 7 members. Examples of heterocycloalkyl groups include, forexample, piperazine, morpholine, piperidine, tetrahydrofuran,pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups includepiperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

The term “heteroaryl” as used herein refers to an aromatic ring systemcontaining at least one heteroatom selected from nitrogen, oxygen, andsulfur. The heteroaryl ring can be fused or otherwise attached to one ormore heteroaryl rings, aromatic or non-aromatic hydrocarbon rings orheterocycloalkyl rings. Examples of heteroaryl groups include, forexample, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline andpyrimidine. Preferred examples of heteroaryl groups include thienyl,benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

The term “C1-C6 hydrocarbyl” as used herein refers to straight,branched, or cyclic alkyl groups having 1-6 carbon atoms, optionallycontaining one or more carbon-carbon double or triple bonds. Examples ofhydrocarbyl groups include, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl andpropargyl. When reference is made herein to C1-C6 hydrocarbyl containingone or two double or triple bonds it is understood that at least twocarbons are present in the alkyl for one double or triple bond, and atleast four carbons for two double or triple bonds.

The term “protecting group” as used herein, refers to groups known inthe art that are readily introduced and removed from an atom, forexample O, N, P, or S. Protecting groups are used to prevent undesirablereactions from taking place that can compete with the formation of aspecific compound or intermediate of interest. See also “ProtectiveGroups in Organic Synthesis”, 3rd Ed., 1999, Greene, T. W. and relatedpublications.

The term “nitrogen protecting group,” as used herein, refers to groupsknown in the art that are readily introduced on to and removed from anitrogen. Examples of nitrogen protecting groups include Boc, Cbz,benzoyl, and benzyl. See also “Protective Groups in Organic Synthesis”,3rd Ed., 1999, Greene, T. W. and related publications.

The term “hydroxy protecting group,” or “hydroxy protection” as usedherein, refers to groups known in the art that are readily introduced onto and removed from an oxygen, specifically an —OH group. Examples ofhyroxy protecting groups include trityl or substituted trityl groups,such as monomethoxytrityl and dimethoxytrityl, or substituted silylgroups, such as tert-butyldimethyl, trimethylsilyl, ortert-butyldiphenyl silyl groups. See also “Protective Groups in OrganicSynthesis”, 3rd Ed., 1999, Greene, T. W. and related publications.

The term “acyl” as used herein refers to —C(O)R groups, wherein R is analkyl or aryl.

The term “phosphorus containing group” as used herein, refers to achemical group containing a phosphorus atom. The phosphorus atom can betrivalent or pentavalent, and can be substituted with O, H, N, S, C orhalogen atoms. Examples of phosphorus containing groups of the instantinvention include but are not limited to phosphorus atoms substitutedwith O, H, N, S, C or halogen atoms, comprising phosphonate,alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramiditegroups, nucleotides and nucleic acid molecules.

The term “phosphine” or “phosphite” as used herein refers to a trivalentphosphorus species, for example compounds having Formula 97:

wherein R can include the groups:

and wherein S and T independently include the groups:

The term “phosphate” as used herein refers to a pentavalent phosphorusspecies, for example a compound having Formula 98:

wherein R includes the groups:

and wherein S and T each independently can be a sulfur or oxygen atom ora group which can include:

and wherein M comprises a sulfur or oxygen atom. The phosphate of theinvention can comprise a nucleotide phosphate, wherein any R, S, or T inFormula 98 comprises a linkage to a nucleic acid or nucleoside.

The term “cationic salt” as used herein refers to any organic orinorganic salt having a net positive charge, for example atriethylammonium (TEA) salt.

The term “degradable linker” as used herein, refers to linker moietiesthat are capable of cleavage under various conditions. Conditionssuitable for cleavage can include but are not limited to pH, UVirradiation, enzymatic activity, temperature, hydrolysis, elimination,and substitution reactions, and thermodynamic properties of the linkage.

The term “photolabile linker” as used herein, refers to linker moietiesas are known in the art, that are selectively cleaved under particularUV wavelengths. Compounds of the invention containing photolabilelinkers can be used to deliver compounds to a target cell or tissue ofinterest, and can be subsequently released in the presence of a UVsource.

The term “nucleic acid conjugates” as used herein, refers to nucleoside,nucleotide and oligonucleotide conjugates.

The term “lipid” as used herein, refers to any lipophilic compound.Non-limiting examples of lipid compounds include fatty acids and theirderivatives, including straight chain, branched chain, saturated andunsaturated fatty acids, carotenoids, terpenes, bile acids, andsteroids, including cholesterol and derivatives or analogs thereof.

The term “folate” as used herein, refers to analogs and derivatives offolic acid, for example antifolates, dihydrofloates, tetrahydrofolates,tetrahydrorpterins, folinic acid, pteropolyglutamic acid, 1-deza,3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza,8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acidderivatives.

The term “compounds with neutral charge” as used herein, refers tocompositions which are neutral or uncharged at neutral or physiologicalpH. Examples of such compounds are cholesterol and other steroids,cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline,distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid,phosphatidic acid and its derivatives, phosphatidyl serine, polyethyleneglycol-conjugated phosphatidylamine, phosphatidylcholine,phosphatidylethanolamine and related variants, prenylated compoundsincluding farnesol, polyprenols, tocopherol, and their modified forms,diacylsuccinyl glycerols, fusogenic or pore forming peptides,dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

The term “lipid aggregate” as used herein refers to a lipid-containingcomposition wherein the lipid is in the form of a liposome, micelle(non-lamellar phase) or other aggregates with one or more lipids.

The term “biological system” as used herein, refers to a eukaryoticsystem or a prokaryotic system, can be a bacterial cell, plant cell or amammalian cell, or can be of plant origin, mammalian origin, yeastorigin, Drosophila origin, or archebacterial origin.

The term “systemic administration” as used herein refers to the in vivosystemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes which lead to systemic absorption include, without limitations:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexpose the desired negatively charged polymers, e.g., nucleic acids, toan accessible diseased tissue. The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, incertain tissue types, such as the tissues of the reticular endothelialsystem (RES). A liposome formulation which can facilitate theassociation of drug with the surface of cells, such as, lymphocytes andmacrophages is also useful. This approach can provide enhanced deliveryof the drug to target cells by taking advantage of the specificity ofmacrophage and lymphocyte immune recognition of abnormal cells, such asthe cancer cells.

The term “pharmacological composition” or “pharmaceutical formulation”refers to a composition or formulation in a form suitable foradministration, for example, systemic administration, into a cell orpatient, preferably a human. Suitable forms, in part, depend upon theuse or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationto reach a target cell (i.e., a cell to which the negatively chargedpolymer is targeted).

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will be first described briefly.

DRAWINGS

FIG. 1 shows examples of chemically stabilized ribozyme motifs. HH Rz,represents hammerhead ribozyme motif (Usman et al., 1996, Curr. Op.Struct. Bio., 1, 527); NCH Rz represents the NCH ribozyme motif (Ludwig& Sproat, International PCT Publication No. WO 98/58058); G-Cleaver,represents G-cleaver ribozyme motif (Kore et al., 1998, Nucleic AcidsResearch 26, 4116-4120, Eckstein et al., International PCT publicationNo. WO 99/16871). N or n, represent independently a nucleotide which canbe same or different and have complementarity to each other; rI,represents ribo-Inosine nucleotide; arrow indicates the site of cleavagewithin the target. Position 4 of the HH Rz and the NCH Rz is shown ashaving 2′-C-allyl modification, but those skilled in the art willrecognize that this position can be modified with other modificationswell known in the art, so long as such modifications do notsignificantly inhibit the activity of the ribozyme.

FIG. 2 shows an example of the Amberzyme ribozyme motif that ischemically stabilized (see for example Beigelman et al., InternationalPCT publication No. WO 99/55857).

FIG. 3 shows an example of the Zinzyme A ribozyme motif that ischemically stabilized (see for example Beigelman et al., Beigelman etal., International PCT publication No. WO 99/55857).

FIG. 4 shows an example of a DNAzyme motif described by Santoro et al.,1997, PNAS, 94, 4262.

FIG. 5 shows a synthetic scheme for the synthesis of a folate conjugateof the instant invention.

FIG. 6 shows representative examples of fludarabine-folate conjugatemolecules of the invention.

FIG. 7 shows a synthetic scheme for post-synthetic modification of anucleic acid molecule to produce a folate conjugate.

FIG. 8 shows a synthetic scheme for generating a protected pteroic acidsynthon of the invention.

FIG. 9 shows a synthetic scheme for generating a 2-dithiopyridylactivated folic acid synthon of the invention.

FIG. 10 shows a synthetic scheme for generating an oligonucleotide ornucleic acid-folate conjugate.

FIG. 11 shows an alternative synthetic scheme for generating anoligonucleotide or nucleic acid-folate conjugate.

FIG. 12 shows an alternative synthetic scheme for post-syntheticmodification of a nucleic acid molecule to produce a folate conjugate.

FIG. 13 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-2′-aminouridine phosphoramiditeconjugate of the invention.

FIG. 14 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-D-threoninol phosphoramiditeconjugate of the invention.

FIG. 15 shows a non-limiting example of a N-acetyl-D-galactosamine siNAnucleic acid conjugate and a N-acetyl-D-galactosamine enzymatic nucleicacid conjugate of the invention. W shown in the example refers to abiodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

FIG. 16 shows a non-limiting example of a synthetic scheme for thesynthesis of a dodecanoic acid derived conjugate linker of theinvention.

FIG. 17 shows a non-limiting example of a synthetic scheme for thesynthesis of an oxime linked nucleic acid/peptide conjugate of theinvention.

FIG. 18 shows non-limiting examples of phospholipid derived nucleic acidconjugates of the invention. W shown in the examples refers to abiodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

FIG. 19 shows a non-limiting example of a synthetic scheme for preparinga phospholipid derived siNA conjugates of the invention.

FIG. 20 shows a non-limiting example of a synthetic scheme for preparinga polyethylene glycol (PEG) derived enzymatic nucleic acid conjugates ofthe invention.

FIG. 21 shows PK data of a 40K PEG conjugated enzymatic nucleic acidmolecule compared to the corresponding non-conjugated enzymatic nucleicacid molecule. The graph is a time course of serum concentration in micedosed with 30 mg/kg of Angiozyme™ or 40-kDa-PEG-Angiozyme™. Thehybridization method was used to quantitate Angiozyme™ levels.

FIG. 22 shows PK data of a phospholipid conjugated enzymatic nucleicacid molecule compared to the corresponding non-conjugated enzymaticnucleic acid molecule.

FIG. 23 shows a non-limiting example of a synthetic scheme for preparinga poly-N-acetyl-D-galactosamine nucleic acid conjugate of the invention.

FIG. 24 a-b shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using oxime and morpholino linkages.

FIG. 25 shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using oxime and phosphoramidate linkages.

FIG. 26 a-b shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using phosphoramidate linkages.

FIG. 27 shows non-limiting examples of phospholipid derivedprotein/peptide conjugates of the invention. W shown in the examplesrefers to a biodegradable linker, for example a nucleic acid dimer,trimer, or tetramer comprising ribonucleotides and/ordeoxyribonucleotides.

FIG. 28 shows a non-limiting example of an N-acetyl-D-galactosaminepeptide/protein conjugate of the invention, the example shown is with apeptide. W shown in the example refers to a biodegradable linker, forexample a nucleic acid dimer, trimer, or tetramer comprisingribonucleotides and/or deoxyribonucleotides.

FIG. 29 shows a non-limiting example of a synthetic approach forsynthesizing peptide or protein conjugates to PEG utilizing abiodegradable linker using phosphoramidate linkages via coupling aprotein phosphoramidite to a PEG conjugated nucleic acid linker.

FIG. 30 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using a phosphoramiditeapproach.

FIG. 31 shows a non-limiting example of the synthesis of siNA PEGconjugates of the invention using NHS ester coupling.

FIG. 32 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using NHS ester coupling.

FIG. 33 shows a non-limiting example of various siNA cholesterolconjugates of the invention.

FIG. 34 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 35 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 36 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 37 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 38 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 39 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 40 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 41 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

FIG. 42 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

FIG. 43 shows a non-limiting example of the pharmacokinetic distributionof intact siNA in liver after administration of conjugated orunconjugated siNA molecules in mice.

FIG. 44 shows a non-limiting example of the activity of conjugated siNAconstructs compared to matched chemistry unconjugated siNA constructs inan HBV cell culture system without the use of transfection lipid. Asshown in the Figure, siNA conjugates provide efficacy in cell culturewithout the need for transfection reagent.

FIG. 45 shows a non-limiting example of a scheme for the synthesis of amono-galactosamine phosphoramidite of the invention that can be used togenerate galactosamine conjugated nucleic acid molecules.

FIG. 46 shows a non-limiting example of a scheme for the synthesis of atri-galactosamine phosphoramidite of the invention that can be used togenerate tri-galactosamine conjugated nucleic acid molecules.

FIG. 47 shows a non-limiting example of a scheme for the synthesis ofanother tri-galactosamine phosphoramidite of the invention that can beused to generate tri-galactosamine conjugated nucleic acid molecules.

FIG. 48 shows a non-limiting example of an alternate scheme for thesynthesis of a tri-galactosamine phosphoramidite of the invention thatcan be used to generate tri-galactosamine conjugated nucleic acidmolecules.

FIG. 49 shows a non-limiting example of a scheme for the synthesis of acholesterol NHS ester of the invention that can be used to generatecholesterol conjugated nucleic acid molecules.

METHOD OF USE

The compositions and conjugates of the instant invention can be used toadminister pharmaceutical agents. Pharmaceutical agents prevent, inhibitthe occurrence, or treat (alleviate a symptom to some extent, preferablyall of the symptoms) of a disease state in a patient.

Generally, the compounds of the instant invention are introduced by anystandard means, with or without stabilizers, buffers, and the like, toform a pharmaceutical composition. For use of a liposome deliverymechanism, standard protocols for formation of liposomes can befollowed. The compositions of the present invention can also beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; sterilesolutions; suspensions for injectable administration; and the like.

The present invention also includes pharmaceutically acceptableformulations of the compounds described above, preferably in combinationwith the molecule(s) to be delivered. These formulations include saltsof the above compounds, e.g., acid addition salts, for example, salts ofhydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.

In one embodiment, the invention features the use of the compounds ofthe invention in a composition comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes). In another embodiment,the invention features the use of compounds of the invention covalentlyattached to polyethylene glycol. These formulations offer a method forincreasing the accumulation of drugs in target tissues. This class ofdrug carriers resists opsonization and elimination by the mononuclearphagocytic system (MPS or RES), thereby enabling longer bloodcirculation times and enhanced tissue exposure for the encapsulated drug(Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem.Pharm. Bull. 1995, 43, 1005-1011). Such compositions have been shown toaccumulate selectively in tumors, presumably by extravasation andcapture in the neovascularized target tissues (Lasic et al., Science1995, 267, 1275-1276; Oku et al., 1995, Biochim. Biophys. Acta, 1238,86-90). The long-circulating compositions enhance the pharmacokineticsand pharmacodynamics of therapeutic compounds, such as DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating compositions are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes a composition(s) prepared forstorage or administration that includes a pharmaceutically effectiveamount of the desired compound(s) in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein. Forexample, preservatives, stabilizers, dyes and flavoring agents can beincluded in the composition. Examples of such agents include but are notlimited to sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. In addition, antioxidants and suspending agents can be included inthe composition.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors which those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer. Furthermore, the compounds ofthe invention and formulations thereof can be administered to a fetusvia administration to the mother of a fetus.

The compounds of the invention and formulations thereof can beadministered orally, topically, parenterally, by inhalation or spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a nucleic acid moleculeof the invention and a pharmaceutically acceptable carrier. One or morenucleic acid molecules of the invention can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients. Thepharmaceutical compositions containing nucleic acid molecules of theinvention can be in a form suitable for oral use, for example, astablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example, sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds of the invention can also be administered in the form ofsuppositories, e.g., for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Compounds of the invention can be administered parenterally in a sterilemedium. The drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anesthetics, preservatives and buffering agentscan be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The compounds of the present invention can also be administered to apatient in combination with other therapeutic compounds to increase theoverall therapeutic effect. The use of multiple compounds to treat anindication can increase the beneficial effects while reducing thepresence of side effects.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small refers to nucleic acid motifs less than about 100 nucleotides inlength, preferably less than about 80 nucleotides in length, and morepreferably less than about 50 nucleotides in length; e.g., antisenseoligonucleotides, hammerhead or the NCH ribozymes) are preferably usedfor exogenous delivery. The simple structure of these moleculesincreases the ability of the nucleic acid to invade targeted regions ofRNA structure. Exemplary molecules of the instant invention arechemically synthesized, and others can similarly be synthesized.

Oligonucleotides (eg; antisense GeneBlocs) are synthesized usingprotocols known in the art as described in Caruthers et al., 1992,Methods in Enzymology 211, 3-19, Thompson et al., International PCTPublication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res.23, 2677-2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennanet al., 1998, Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No.6,001,311. All of these references are incorporated herein by reference.The synthesis of oligonucleotides makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. In a non-limiting example, smallscale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer using a 0.2 μmol scale protocol with a 2.5 min coupling stepfor 2′-O-methylated nucleotides and a 45 sec coupling step for 2′-deoxynucleotides. Table II outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. In a non-limiting example, a 33-fold excess(60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a 105-foldexcess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can be used ineach coupling cycle of 2′-O-methyl residues relative to polymer-bound5′-hydroxyl. In a non-limiting example, a 22-fold excess (40 μL of 0.11M=4.4 μmol) of deoxy phosphoramidite and a 70-fold excess of S-ethyltetrazole (40 μL of 0.25 M=10 μmol) can be used in each coupling cycleof deoxy residues relative to polymer-bound 5′-hydroxyl. Averagecoupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer includebut are not limited to;detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the antisense oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aq. methylamine(1 mL) at 65° C. for 10 min. After cooling to −20° C., the supernatantis removed from the polymer support. The support is washed three timeswith 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is thenadded to the first supernatant. The combined supernatants, containingthe oligoribonucleotide, are dried to a white powder. Standard drying orlyophilization methods known to those skilled in the art can be used.

The method of synthesis used for normal RNA including certain enzymaticnucleic acid molecules follows the procedure as described in Usman etal., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, NucleicAcids Res., 18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23,2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes useof common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 7.5 min coupling step for alkylsilyl protected nucleotides and a2.5 min coupling step for 2′-O-methylated nucleotides. Table II outlinesthe amounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include; detritylation solution is3% TCA in methylene chloride (ABI); capping is performed with 16%N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM I₂, 49 mMpyridine, 9% water in THF (PERSEPTIVE™). Burdick & Jackson SynthesisGrade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 min. The vial is brought to r.t. TEA.3HF (0.1 mL) isadded and the vial is heated at 65° C. for 15 min. The sample is cooledat −20° C. and then quenched with 1.5 M NH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 min. The cartridge is then washed again with water, salt exchangedwith 1 M NaCl and washed with water again. The oligonucleotide is theneluted with 30% acetonitrile.

Inactive hammerhead ribozymes or binding attenuated control ((BAC)oligonucleotides) are synthesized by substituting a U for G₅ and a U forA₁₄ (numbering from Hertel, K. J., et al., 1992, Nucleic Acids Res., 20,3252). Similarly, one or more nucleotide substitutions can be introducedin other enzymatic nucleic acid molecules to inactivate the molecule andsuch molecules can serve as a negative control.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including, but notlimited to, 96 well format, with the ratio of chemicals used in thereaction being adjusted accordingly.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204).

The nucleic acid molecules of the present invention are modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). Nucleic acid conjugates of theinvention can purified by gel electrophoresis using general methods orare purified by high pressure liquid chromatography (HPLC; See Wincottet al., Supra, the totality of which is hereby incorporated herein byreference) or hydrophobic interaction chromatography and arere-suspended in water.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) that prevent their degradation by serumribonucleases can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al.,supra; all of these describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules herein). Modifications which enhance their efficacy in cells,and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; allof the references are hereby incorporated in their totality by referenceherein). Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into ribozymes without inhibiting catalysis,and are incorporated by reference herein. In view of such teachings,similar modifications can be used as described herein to modify thenucleic acid molecules of the instant invention.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorothioate, and/or 5′-methylphosphonatelinkages improves stability, too many of these modifications may causesome toxicity. Therefore, when designing nucleic acid molecules theamount of these internucleotide linkages should be minimized. Withoutbeing bound by any particular theory, the reduction in the concentrationof these linkages should lower toxicity resulting in increased efficacyand higher specificity of these molecules.

Nucleic acid molecules having chemical modifications that maintain orenhance activity are provided. Such nucleic acid is also generally moreresistant to nucleases than unmodified nucleic acid. Thus, in a celland/or in vivo the activity can not be significantly lowered.Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acidmolecules and antisense nucleic acid molecules) delivered exogenouslyare optimally stable within cells until translation of the target RNAhas been inhibited long enough to reduce the levels of the undesirableprotein. This period of time varies between hours to days depending uponthe disease state. The nucleic acid molecules should be resistant tonucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of RNA and DNA (Wincottet al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,Methods in Enzymology 211, 3-19 (incorporated by reference herein) haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

Use of the nucleic acid-based molecules of the invention can lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple antisense or enzymatic nucleicacid molecules targeted to different genes, nucleic acid moleculescoupled with known small molecule inhibitors, or intermittent treatmentwith combinations of molecules (including different motifs) and/or otherchemical or biological molecules). The treatment of patients withnucleic acid molecules can also include combinations of different typesof nucleic acid molecules.

In another embodiment, nucleic acid catalysts having chemicalmodifications that maintain or enhance enzymatic activity are provided.Such nucleic acids are also generally more resistant to nucleases thanunmodified nucleic acid. Thus, in a cell and/or in vivo the activity ofthe nucleic acid can not be significantly lowered. As exemplified hereinsuch enzymatic nucleic acids are useful in a cell and/or in vivo even ifactivity over all is reduced 10 fold (Burgin et al., 1996, Biochemistry,35, 14090). Such enzymatic nucleic acids herein are said to “maintain”the enzymatic activity of an all RNA ribozyme or all DNA DNAzyme.

In another aspect the nucleic acid molecules comprise a 5′ and/or a3′-cap structure.

In another embodiment the 3′-cap includes, for example 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

In one embodiment, the invention features modified enzymatic nucleicacid molecules with phosphate backbone modifications comprising one ormore phosphorothioate, phosphorodithioate, methylphosphonate,morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide,sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/oralkylsilyl, substitutions. For a review of oligonucleotide backbonemodifications see Hunziker and Leumann, 1995, Nucleic Acid Analogues:Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417, andMesmaeker et al., 1994, Novel Backbone Replacements forOligonucleotides, in Carbohydrate Modifications in Antisense Research,ACS, 24-39. These references are hereby incorporated by referenceherein.

In connection with 2′-modified nucleotides as described for theinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO98/28317, respectively, which are both incorporated by reference intheir entireties.

Various modifications to nucleic acid (e.g., antisense and ribozyme)structure can be made to enhance the utility of these molecules. Forexample, such modifications can enhance shelf-life, half-life in vitro,stability, and ease of introduction of such oligonucleotides to thetarget site, including e.g., enhancing penetration of cellular membranesand conferring the ability to recognize and bind to targeted cells.

Use of these molecules can lead to better treatment of diseaseprogression by affording the possibility of combination therapies (e.g.,multiple enzymatic nucleic acid molecules targeted to different genes,enzymatic nucleic acid molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations of enzymaticnucleic acid molecules (including different enzymatic nucleic acidmolecule motifs) and/or other chemical or biological molecules). Thetreatment of patients with nucleic acid molecules can also includecombinations of different types of nucleic acid molecules. Therapies canbe devised which include a mixture of enzymatic nucleic acid molecules(including different enzymatic nucleic acid molecule motifs), antisenseand/or 2-5A chimera molecules to one or more targets to alleviatesymptoms of a disease.

Indications

Particular disease states that can be treated using compounds andcompositions of the invention include, but are not limited to, cancersand cancerous conditions such as breast, lung, prostate, colorectal,brain, esophageal, stomach, bladder, pancreatic, cervical,hepatocellular, head and neck, and ovarian cancer, melanoma, lymphoma,glioma, multidrug resistant cancers; ocular conditions such as maculardegeneration and diabetic retinopathy, and/or viral infections includingHIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus, westnile virus, severe acute respiratory syndrome (SARS) virus, Ebola virus,foot and mouth virus, and papilloma virus infection.

The molecules of the invention can be used in conjunction with otherknown methods, therapies, or drugs. For example, the use of monoclonalantibodies (eg; mAb IMC C225, mAB ABX-EGF) treatment, tyrosine kinaseinhibitors (TKIs), for example OSI-774 and ZD1839, chemotherapy, and/orradiation therapy, are all non-limiting examples of a methods that canbe combined with or used in conjunction with the compounds of theinstant invention. Common chemotherapies that can be combined withnucleic acid molecules of the instant invention include variouscombinations of cytotoxic drugs to kill the cancer cells. These drugsinclude, but are not limited to, paclitaxel (Taxol), docetaxel,cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracilcarboplatin, edatrexate, gemcitabine, vinorelbine etc. Those skilled inthe art will recognize that other drug compounds and therapies can besimilarly be readily combined with the compounds of the instantinvention are hence within the scope of the instant invention.

Diagnostic Uses

The compounds of this invention, for example, nucleic acid conjugatemolecules, can be used as diagnostic tools to examine genetic drift andmutations within diseased cells or to detect the presence of a diseaserelated RNA in a cell. The close relationship between, for example,enzymatic nucleic acid molecule activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple enzymatic nucleic acid moleculesconjugates of the invention, one can map nucleotide changes which areimportant to RNA structure and function in vitro, as well as in cellsand tissues. Cleavage of target RNAs with enzymatic nucleic acidmolecules can be used to inhibit gene expression and define the role(essentially) of specified gene products in the progression of disease.In this manner, other genetic targets can be defined as importantmediators of the disease. These experiments can lead to better treatmentof the disease progression by affording the possibility of combinationaltherapies (e.g., multiple enzymatic nucleic acid molecules targeted todifferent genes, enzymatic nucleic acid molecules coupled with knownsmall molecule inhibitors, or intermittent treatment with combinationsof enzymatic nucleic acid molecules and/or other chemical or biologicalmolecules). Other in vitro uses of enzymatic nucleic acid molecules ofthis invention are well known in the art, and include detection of thepresence of mRNAs associated with a disease-related condition. Such RNAis detected by determining the presence of a cleavage product aftertreatment with an enzymatic nucleic acid molecule using standardmethodology.

In a specific example, enzymatic nucleic acid molecules that aredelivered to cells as conjugates and which cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstenzymatic nucleic acid molecule is used to identify wild-type RNApresent in the sample and the second enzymatic nucleic acid molecule isused to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth enzymatic nucleic acid molecules to demonstrate the relativeenzymatic nucleic acid molecule efficiencies in the reactions and theabsence of cleavage of the “non-targeted” RNA species. The cleavageproducts from the synthetic substrates also serve to generate sizemarkers for the analysis of wild-type and mutant RNAs in the samplepopulation. Thus each analysis requires two enzymatic nucleic acidmolecules, two substrates and one unknown sample which is combined intosix reactions. The presence of cleavage products is determined using anRNAse protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype is adequate to establishrisk. If probes of comparable specific activity are used for bothtranscripts, then a qualitative comparison of RNA levels will beadequate and will decrease the cost of the initial diagnosis. Highermutant form to wild-type ratios are correlated with higher risk whetherRNA levels are compared qualitatively or quantitatively. The use ofenzymatic nucleic acid molecules in diagnostic applications contemplatedby the instant invention is more fully described in George et al., U.S.Pat. Nos. 5,834,186 and 5,741,679, Shih et al., U.S. Pat. No. 5,589,332,Nathan et al., U.S. Pat. No. 5,871,914, Nathan and Ellington,International PCT publication No. WO 00/24931, Breaker et al.,International PCT Publication Nos. WO 00/26226 and 98/27104, andSullenger et al., International PCT publication No. WO 99/29842.

Additional Uses

Potential uses of sequence-specific enzymatic nucleic acid molecules ofthe instant invention that are delivered to cells as conjugates can havemany of the same applications for the study of RNA that DNA restrictionendonucleases have for the study of DNA (Nathans et al., 1975 Ann. Rev.Biochem. 44:273). For example, the pattern of restriction fragments canbe used to establish sequence relationships between two related RNAs,and large RNAs can be specifically cleaved to fragments of a size moreuseful for study. The ability to engineer sequence specificity of theenzymatic nucleic acid molecule is ideal for cleavage of RNAs of unknownsequence. Applicant has described the use of nucleic acid molecules todown-regulate gene expression of target genes in bacterial, microbial,fungal, viral, and eukaryotic systems including plant, or mammaliancells.

Example 1 Synthesis ofO¹-(4-monomethoxytrityl)-N-(6-(N-(α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate 3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20)(FIG. 5)

General. All reactions were carried out under a positive pressure ofargon in anhydrous solvents. Commercially available reagents andanhydrous solvents were used without further purification. ¹H (400.035MHz) and ³¹P (161.947 MHz) NMR spectra were recorded in CDCl₃, unlessstated otherwise, and chemical shifts in ppm refer to TMS and H₃PO₄,respectively. Analytical thin-layer chromatography (TLC) was performedwith Merck Art.5554 Kieselgel 60 F₂₅₄ plates and flash columnchromatography using Merck 0.040-0.063 mm silica gel 60.

N—(N-Fmoc-6-aminocaproyl)-D-threoninol (13). N-Fmoc-6-aminocaproic acid(10 g, 28.30 mmol) was dissolved in DMF (50 ml) and N-hydroxysuccinimide(3.26 g, 28.30 mmol) and 1,3-dicyclohexylcarbodiimide (5.84 g, 28.3mmol) were added to the solution. The reaction mixture was stirred at RT(about 23° C.) overnight and the precipitated 1,3-dicyclohexylureafiltered off. To the filtrate D-threoninol (2.98 g, 28.30 mmol) wasadded and the reaction mixture stirred at RT overnight. The solution wasreduced to ca half the volume in vacuo, the residue diluted with about mml of ethyl acetate and extracted with about x ml of 5% NaHCO₃, followedby washing with brine. The organic layer was dried (Na₂SO₄), evaporatedto a syrup and chromatographed by silica gel column chromatography using1-10% gradient of methanol in ethyl acetate. Fractions containing theproduct were pooled and evaporated to a white solid (9.94 g, 80%).¹H-NMR (DMSO-d₆-D₂O)

7.97-7.30 (m, 8H, aromatic), 4.34 (d, J=6.80, 2H, Fm), 4.26 (t, J=6.80,1H, Fm), 3.9 (m, 1H, H3 Thr), 3.69 (m, 1H, H2 Thr), 3.49 (dd, J=10.6,J=7.0, 1H, H1 Thr), 3.35 (dd, J=10.6, J=6.2, 1H, H1′ Thr), 3.01 (m, 2H,CH₂CO Acp), 2.17 (m, 2H, CH₂NH Acp), 1.54 (m, 2H, CH₂ Acp), 1.45 (m, 2H,CH₂ Acp), 1.27 (m, 2H, CH₂ Acp), 1.04 (d, J=6.4, 3H, CH₃). MS/ESI⁺ m/z441.0 (M+H)⁺.

O¹-(4-Monomethoxytrityl)-N—(N-Fmoc-6-aminocaproyl)-D-threoninol (14). Tothe solution of 13 (6 g, 13.62 mmol) in dry pyridine (80 ml)p-anisylchlorodiphenyl-methane (6 g, 19.43 mmol) was added and thereaction mixture stirred at RT overnight. Methanol was added (20 ml) andthe solution concentrated in vacuo. The residual syrup was partitionedbetween about x ml of dichloromethane and about x ml of 5% NaHCO₃, theorganic layer was washed with brine, dried (Na₂SO₄) and evaporated todryness. Flash column chromatography using 1-3% gradient of methanol indichloromethane afforded 14 as a white foam (6 g, 62%). ¹H-NMR (DMSO)

7.97-6.94 (m, 22H, aromatic), 4.58 (d, 1H, J=5.2, OH), 4.35 (d, J=6.8,2H, Fm), 4.27 (t, J=6.8, 1H, Fm), 3.97 (m, 2H, H2, H3 Thr), 3.80 (s, 3H,OCH₃), 3.13 (dd, J=8.4, J=5.6, 1H, H1 Thr), 3.01 (m, 2H, CH₂CO Acp),2.92 (m, dd, J=8.4, J=6.4, 1H, H1′ Thr), 2.21 (m, 2H, CH₂NH Acp), 1.57(m, 2H, CH₂ Acp), 1.46 (m, 2H, CH₂ Acp), 1.30 (m, 2H, CH₂ Acp), 1.02 (d,J=5.6, 3H, CH₃). MS/ESI⁺ m/z 735.5 (M+Na)⁺.

O¹-(4-Monomethoxytrityl)-N-(6-aminocaproyl)-D-threoninol (15). 14 (9.1g, 12.77 mmol) was dissolved in DMF (100 ml) containing piperidine (10ml) and the reaction mixture was kept at RT for about 1 hour. Thesolvents were removed in vacuo and the residue purified by silica gelcolumn chromatography using 1-10% gradient of methanol indichloromethane to afford 15 as a syrup (4.46 g, 71%). ¹H-NMR

7.48-6.92 (m, 14H, aromatic), 6.16 (d, J=8.8, 1H, NH), 4.17 (m, 1H, H3Thr), 4.02 (m, 1H, H2 Thr), 3.86 (s, 3H, OCH₃), 3.50 (dd, J=9.7, J=4.4,1H, H1 Thr), 3.37 (dd, J=9.7, J=3.4, 1H, H1′ Thr), 2.78 (t, J=6.8, 2H,CH₂CO Acp), 2.33 (t, J=7.6, 2H, CH₂NH Acp), 1.76 (m, 2H, CH₂ Acp), 1.56(m, 2H, CH₂ Acp), 1.50 (m, 2H, CH₂ Acp), 1.21 (d, J=6.4, 3H, CH₃).MS/ESI⁺ m/z 491.5 (M+H)⁺.

O¹-(4-Monomethoxytrityl)-N-(6-(N—(N-Boc-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol(16). To the solution of N-Boc-α-OFm-glutamic acid (Bachem) (1.91 g,4.48 mmol) in DMF (10 ml) N-hydroxysuccinimide (518 mg, 4.50 mmol) and1,3-dicyclohexylcarbodiimide (928 mg, 4.50 mmol) was added and thereaction mixture was stirred at RT overnight. 1,3-Dicyclohexylurea wasfiltered off and to the filtrate 15 (2 g, 4.08 mmol) and pyridine (2 ml)were added. The reaction mixture was stirred at RT for 3 hours and thanconcentrated in vacuo. The residue was partitioned between ethyl acetateand 5% Na₂HCO₃, the organic layer extracted with brine as previouslydescribed, dried (Na₂SO₄) and evaporated to a syrup. Columnchromatography using 2-10% gradient of methanol in dichlotomethaneafforded 16 as a white foam (3.4 g, 93%). ¹H-NMR

7.86-6.91 (m, 22H, aromatic), 6.13 (d, J=8.8, 1H, NH), 5.93 (br s, 1H,NH), 5.43 (d, J=8.4, 1H, NH), 4.63 (dd, J=10.6, J=6.4, 1H, Fm), 4.54(dd, J=10.6, J=6.4, 1H, Fm), 4.38 (m, 1H, Glu), 4.3 (t, J=6.4, 1H, Fm),4.18 (m, 1H, H3 Thr), 4.01 (m, 1H, H2 Thr), 3.88 (s, 3H, OCH₃), 3.49(dd, J=9.5, J=4.4, 1H, H1 Thr), 3.37 (dd, J=9.5, J=3.8, 1H, H1′ Thr),3.32 (m, 2H, CH₂CO Acp), 3.09 (br s, 1H, OH), 2.32 (m, 2H, CH₂NH Acp),2.17 (m, 3H, Glu), 1.97 (m, 1H, Glu), 1.77 (m, 2H, CH₂ Acp), 1.61 (m,2H, CH₂ Acp), 1.52 (s, 9H, t-Bu), 1.21 (d, J=6.4, 3H, CH₃). MS/ESI⁺ m/z920.5 (M+Na)⁺.

N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol hydrochloride (17).16 (2 g, 2.23 mmol) was dissolved in methanol (30 ml) containing anisole(10 ml) and to this solution x ml of 4M HCl in dioxane was added. Thereaction mixture was stirred for 3 hours at RT and then concentrated invacuo. The residue was dissolved in ethanol and the product precipitatedby addition of x ml of ether. The precipitate was washed with ether anddried to give 17 as a colorless foam (1 g, 80%). ¹H-NMR (DMSO-d₆-D₂O)

7.97-7.40 (m, 8H, aromatic), 4.70 (m, 1H, Fm), 4.55 (m, 1H, Fm), 4.40(t, J=6.4, 1H, Fm), 4.14 (t, J=6.6, 1H, Glu), 3.90 (dd, J=2.8, J=6.4,1H, H3 Thr), 3.68 (m, 1H, H2 Thr), 3.49 (dd, J=10.6, J=7.0, 1H, H1 Thr),3.36 (dd, J=10.6, J=6.2, 1H, H1′ Thr), 3.07 (m, 2H, CH₂CO Acp), 2.17 m,3H), 1.93 (m, 2H), 1.45 (m, 2H), 1.27 (m, 2H), 1.04 (d, J=6.4, 3H Thr).MS/ESI⁺ m/z 526.5 (M+H)⁺.

N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (18). To the solution of N²-iBu-N¹⁰-TFA-pteroic acid¹(480 mg, 1 mmol) in DMF (5 ml) 1-hydroxybenzotriazole (203 mg, 1.50mmol), EDCI (288 mg, 1.50 mmol) and 17 (free base, 631 mg, 1.2 mmol) areadded. The reaction mixture is stirred at RT for 2 hours, thenconcentrated to ca 3 ml and loaded on the column of silica gel. Elutionwith dichloromethane, followed by 1-20% gradient of methanol indichloromethane afforded 18 (0.5 g, 51%). ¹H-NMR (DMSO-d₆-D₂O) δ 9.09(d, J=6.8, 1H, NH) 8.96 (s, 1H, H7 pteroic acid), 8.02-7.19 (m, 13H,aromatic, NH), 5.30 (s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.41 (d,J=6.8, 2H, Fm), 4.29 (t, J=6.8, 1H, Fm), 3.89 (dd, J=6.2, J=2.8, 1H, H3Thr), 3.68 (m, 1H, H2 Thr), 3.48 (dd, J=10.4, J=7.0, 1H, H1 Thr), 3.36(dd, J=10.4, J=6.2, 1H H1′ Thr), 3.06 (m, 2H, CH₂CO Acp), 2.84 (m, 1H,iBu), 2.25 (m, 2H, CH₂NH Acp), 2.16 (m, 3H, Glu), 1.99 (m, 1H, Glu),1.52 (m, 2H Acp), 1.42 (m, 2H Acp), 1.27 (m, 2H Acp), 1.20 (s, 3H iBu),1.19 (s, 3H, iBu), 1.03 (d, J=6.2, 3H Thr). MS/ESI⁻ m/z 984.5 (M−H)⁻.

O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate (19). To the solution of conjugate 18 (1 g, 1.01 mmol) indry pyridine (15 ml) p-anisylchlorodiphenylmethane (405 mg) was addedand the reaction mixture was stirred, protected from moisture, at RTovernight. Methanol (3 ml) was added and the reaction mixtureconcentrated to a syrup in vacuo. The residue was partitioned betweendichloromethane and 5% NaHCO₃, the organic layer washed with brine,dried (Na₂SO₄) and evaporated to dryness. Column chromatography using0.5-10% gradient of methanol in dichloromethane afforded 19 as acolorless foam (0.5 g, 39%. ¹H-NMR (DMSO-d₆-D₂O δ9.09 (d, J=6.8, 1H, NH)8.94 (s, 1H, H7 pteroic acid), 8.00-6.93 (m, 27H, aromatic, NH), 5.30(s, 2H, pteroic acid), 4.50 (m, 1H, Glu), 4.40 (d, J=6.8, 2H, Fm), 4.29(t, J=6.8, 1H, Fm), 3.94 (m, 2H, H3, H2 Thr), 3.79 (s, 3H, OCH₃) 3.11(dd, J=8.6, J=5.8, 1H, H1 Thr), 3.04 (m, 2H, CH₂CO Acp), 2.91 (dd,J=8.6, J=6.4, 1H, H1′ Thr), 2.85 (m, 1H, iBu), 2.25 (m, 2H, CH₂NH Acp),2.19 (m, 2H, Glu), 2.13 (m, 1H, Glu), 1.98 (m, 1H, Glu), 1.55 (m, 2HAcp), 1.42 (m, 2H Acp), 1.29 (m, 2H Acp), 1.20 (s, 3H iBu), 1.18 (s, 3H,iBu), 1.00 (d, J=6.4, 3H Thr). MS/ESI⁻ m/z 1257.0 (M−H)⁻.

O¹-(4-monomethoxytrityl)-N-(6-(N-α-OFm-L-glutamyl)aminocaproyl))-D-threoninol-N²-iBu-N¹⁰-TFA-pteroicacid conjugate 3′-O-(2-cyanoethyl-N,N-diisopropylphosphor-amidite) (20).To the solution of 19 (500 mg, 0.40 mmol) in dichloromethane (2 ml)2-cyanoethyl tetraisopropylphosphordiamidite (152 μL, 0.48 mmol) wasadded followed by pyridinium trifluoroacetate (93 mg, 0.48 mmol). Thereaction mixture was stirred at RT for 1 hour and than loaded on thecolumn of silica gel in hexanes. Elution using ethyl acetate-hexanes1:1, followed by ethyl acetate and ethyl acetate-acetone 1:1 in thepresence of 1% pyridine afforded 20 as a colorless foam (480 mg, 83%).³¹P NMR δ 149.4 (s), 149.0 (s).

Example 2 Synthesis of 2-dithiopyridyl activated folic acid (30) (FIG.9)

Synthesis of the cysteamine modified folate 30 is presented in FIG. 9.Monomethoxytrityl cysteamine 21 was prepared by selective tritylation ofthe thiol group of cysteamine with 4-methoxytrityl alcohol intrifluoroacetic acid. Peptide coupling of 21 with Fmoc-Glu-OtBu (BachemBioscience Inc., King of Prussia, Pa.) in the presence of PyBOP yielded22 in a high yield. N-Fmoc group was removed smoothly with piperidine togive 23. Condensation of 23 with p-(4-methoxytrityl)aminobenzoic acid,prepared by reaction of p-aminobenzoic acid with 4-methoxytritylchloride in pyridine, afforded the fully protected conjugate 24.Selective cleavage of N-MMTr group with acetic acid afforded 25 inquantitative yield. Shiff base formation between 25 andN²-iBu-6-formylpterin 26,⁹ followed by reduction with borane-pyridinecomplex proceeded with a good yield to give fully protectedcysteamine-folate adduct 27.¹² The consecutive cleavage of protectinggroups of 27 with base and acid yielded thiol derivative 29. The thiolexchange reaction of 29 with 2,2-dipyridyl disulfide afforded thedesired S-pyridyl activated synthon 30 as a yellow powder; Isolated as aTEA⁺+salt: ¹H NMR spectrum for 10 in D₂O: δ8.68 (s, 1H, H-7), 8.10 (d,J=3.6, 1H, pyr), 7.61 (d, J=8.8, 2H, PABA), 7.43 (m, 1H, pyr), 7.04 (d,J=7.6, 1H, pyr), 6.93 (m, 1H, pyr), 6.82 (d, J=8.8, 1H, PABA), 4.60 (s,2H, 6-CH₂), 4.28 (m, 1H, Glu), 3.30-3.08 (m, 2H, cysteamine), 3.05 (m,6H, TEA), 2.37 (m, 2H, cysteamine), 2.10 (m, 4H, Glu), 1.20 (m, 9H,TEA). MS/ESI⁻ m/z 608.02 [M−H]⁻. It is worth noting that the isolationof 30 as its TEA⁺ or Na⁺ salt made it soluble in DMSO and/or water,which is an important requirement for its use in conjugation reactions.

Example 3 Post Synthetic Conjugation of Enzymatic Nucleic Acid to FormNucleic Acid-Folate Conjugate (33) (FIG. 10)

Oligonucleotide synthesis, deprotection and purification was performedas described herein. 5′-Thiol-Modifier C6 (Glen Research, Sterling, Va.)was coupled as the last phosphoramidite to the 5′-end of a growingoligonucleotide chain. After cleavage from the solid support and basedeprotection, the disulfide modified enzymatic nucleic acid molecule 31(FIG. 10) was purified using ion exchange chromatography. The thiolgroup was unmasked by reduction with dithiothreitol (DTT) to afford 32which was purified by gel filtration and immediately conjugated with 30.The resulting conjugate 33 was separated from the excess folate by gelfiltration and then purified by RP HPLC using gradient of acetonitrilein 50 mM triethylammonium acetate (TEAA). Desalting was performed by RPHPLC. Reactions were conducted on 400 mg of disulfide modified enzymaticnucleic acid molecule 31 to afford 200-250 mg (50-60% yield) ofconjugate 33. MALDI TOF MS confirmed the structure: 13 [M−H]⁻ 12084.74(calc. 12083.82). An alternative approach to this synthesis is shown inFIG. 11.

As shown in Examples 2 and 3, a folate-cysteamine adduct can be preparedby a scaleable solution phase synthesis in a good overall yield.Disulfide conjugation of this novel targeting ligand to thethiol-modified oligonucleotide is suitable for the multi-gram scalesynthesis. The 9-atom spacer provides a useful spatial separationbetween folate and attached oligonucleotide cargo. Importantly,conjugation of folate to the oligonucleotide through a disulfide bondshould permit intermolecular separation which was suggested to berequired for the functional cytosolic entry of a protein drug.

Example 4 Synthesis of Galactose and N-acetyl-Galactosamine Conjugates(FIGS. 13, 14, and 15)

Applicant has designed both nucleoside andnon-nucleoside-N-acetyl-D-galactosamine conjugates suitable forincorporation at any desired position of an oligonucleotide. Multipleincorporations of these monomers could result in a “glycoside clustereffect”.

All reactions were carried out under a positive pressure of argon inanhydrous solvents. Commercially available reagents and anhydroussolvents were used without further purification.N-acetyl-D-galactosamine was purchased from Pfanstiel (Waukegan, Ill.),folic acid from Sigma (St. Louis, Mo.), D-threoninol from Aldrich(Milwaukee, Wis.) and N-Boc-α-OFm glutamic acid from Bachem. ¹H (400.035MHz) and ³¹P (161.947 MHz) NMR spectra were recorded in CDCl₃, unlessstated otherwise, and chemical shifts in ppm refer to TMS and H3PO4,respectively. Analytical thin-layer chromatography (TLC) was performedwith Merck Art.5554 Kieselgel 60 F₂₅₄ plates and flash columnchromatography using Merck 0.040-0.063 mm silica gel 60. The generalprocedures for RNA synthesis, deprotection and purification aredescribed herein. MALDI-TOF mass spectra were determined on PerSeptiveBiosystems Voyager spectrometer. Electrospray mass spectrometry was runon the PE/Sciex API365 instrument.

2′-(N-L-lysyl)amino-5′-O-4,4′-dimethoxytrityl-2′-deoxyuridine (2).2′-(N-α,ε-bis-Fmoc-L-lysyl)amino-5′-O-4,4′-dimethoxytrityl-2′-deoxyuridine(1) (4 g, 3.58 mmol) was dissolved in anhydrous DMF (30 ml) anddiethylamine (4 ml) was added. The reaction mixture was stirred at rtfor 5 hours and than concentrated (oil pump) to a syrup. The residue wasdissolved in ethanol and ether was added to precipitate the product (1.8g, 75%). ¹H-NMR (DMSO-d₆-D₂O) _(δ) 7.70 (d, _(J) _(6,5) =8.4, 1H, H6),7.48-6.95 (m, 13H, aromatic), 5.93 (d, J_(1′,2′)=8.4, 1H, H1′), 5.41 (d,J_(5,6)=8.4, 1H, H5), 4.62 (m, 1H, H2′), 4.19 (d, 1H, _(J) _(3′,2′)=6.0, H3′), 3.81 (s, 6H, 2×OMe), 3.30 (m, 4H, 2H5′, CH₂), 1.60-1.20 (m,6H, 3×CH₂). MS/ESI⁺ m/z 674.0 (M+H)⁺.

N-Acetyl-1,4,6-tri-O-acetyl-2-amino-2-deoxy-β-D-galactospyranose (3).N-Acetyl-D-galac-tosamine (6.77 g, 30.60 mmol) was suspended inacetonitrile (200 ml) and triethylamine (50 ml, 359 mmol) was added. Themixture was cooled in an ice-bath and acetic anhydride (50 ml, 530mmol)) was added dropwise under cooling. The suspension slowly clearedand was then stirred at rt for 2 hours. It was than cooled in anice-bath and methanol (60 ml) was added and the stirring continued for15 min. The mixture was concentrated under reduced pressure and theresidue partitioned between dichloromethane and 1 N HCl. Organic layerwas washed twice with 5% NaHCO₃, followed by brine, dried (Na₂SO₄) andevaporated to dryness to afford 10 g (84%) of 3 as a colorless foam. ¹HNMR was in agreement with published data (Findeis, 1994, Int. J. PeptideProtein Res., 43, 477-485.

2-Acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactospyranose (4). Thiscompound was prepared from 3 as described by Findeis supra.

Benzyl 12-Hydroxydodecanoate (5). To a cooled (0° C.) and stirredsolution of 12-hydroxydodecanoic acid (10.65 g, 49.2 mmol) in DMF (70ml) DBU (8.2 ml, 54.1 mmol) was added, followed by benzyl bromide (6.44ml, 54.1 mmol). The mixture was left overnight at rt, than concentratedunder reduced pressure and partitioned between 1 N HCl and ether.Organic phase was washed with saturated NaHCO₃, dried over Na₂SO₄ andevaporated. Flash chromatography using 20-30% gradient of ethyl acetatein hexanes afforded benzyl ester as a white powder (14.1 g, 93.4%).¹H-NMR spectral data were in accordance with the published values.³³

12′-Benzylhydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyrano-se(6). 1-Chloro sugar 4 (4.26 g, 11.67 mmol) and benzyl12-hydroxydodecanoate (5) (4.3 g, 13.03 mmol) were dissolved innitromethane-toluene 1:1 (122 ml) under argon and Hg(CN)₂ (3.51 g, 13.89mmol) and powdered molecular sieves 4A (1.26 g) were added. The mixturewas stirred at rt for 24 h, filtered and the filtrate concentrated underreduced pressure. The residue was partitioned between dichloromethaneand brine, organic layer was washed with brine, followed by 0.5 M KBr,dried (Na₂SO₄) and evaporated to a syrup. Flash silica gel columnchromatography using 15-30% gradient of acetone in hexanes yieldedproduct 6 as a colorless foam (6 g, 81%). ¹H-NMR _(δ) 7.43 (m, _(5H),phenyl), 5.60 (d, 1H, J_(NH,2)=8.8, NH), 5.44 (d, J_(4,3)=3.2, 1H, H4),5.40 (dd, _(J3,4)=3.2, J_(3,2)=10.8, 1H, H3), 5.19 (s, 2H, CH₂Ph), 4.80(d, J_(1,2)=8.0, 1H, H1), 4.23 (m, 2H, CH₂), 3.99 (m, 3H, H2, H6), 3.56(m, 1H, H5), 2.43 (t, J=7.2, 2H, CH₂), 2.22 (s, 3H, Ac), 2.12 (s, 3H,Ac), 2.08 (s, 3H, Ac), 2.03 (s, 3H, Ac), 1.64 (m, 4H, 2×CH₂), 1.33 (brm, 14H, 7×CH₂). MS/ESI⁻ m/z 634.5 (M−H)⁻.

12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose(7).

Conjugate 6 (2 g, 3.14 mmol)) was dissolved in ethanol (50 ml) and 5%Pd—C (0.3 g) was added. The reaction mixture was hydrogenated overnightat 45 psi H₂, the catalyst was filtered off and the filtrate evaporatedto dryness to afford pure 7 (1.7 g, quantitative) as a white foam.¹H-NMR _(δ) 5.73 (d, 1H, J_(NH,2)=8.4, NH), 5.44 (d, J_(4,3)=3.0, 1H,H4), 5.40 (dd, J_(3,4)=3.0, J_(3,2)=11.2, 1H, H3), 4.78 (d, J_(1,2)=8.8,1H, H1), 4.21 (m, 2H, CH₂), 4.02 (m, 3H, H2, H6), 3.55 (m, 1H, H5), 2.42(m, 2H, CH₂), 2.23 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.04(s, 3H, Ac), 1.69 (m, 4H, 2×CH₂), 1.36 (br m, 14H, 7×CH₂). MS/ESI⁻ m/z544.0 (M−H)⁻.

2′-(N-α-bis-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose)-L-lysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityluridine (9). 7 (1.05 g, 1.92 mmol) was dissolved in anhydrous THF andN-hydroxysuccinimide (0.27 g, 2.35 mmol) and1,3-dicyclohexylcarbodiimide (0.55 g, 2.67 mmol) were added. Thereaction mixture was stirred at rt overnight, then filtered throughCelite pad and the filtrate concentrated under reduced pressure. Thecrude NHSu ester 8 was dissolved in dry DMF (13 ml) containingdiisopropylethylamine (0.67 ml, 3.85 mmol) and to this solutionnucleoside 2 (0.64 g, 0.95 mmol was added). The reaction mixture wasstirred at rt overnight and than concentrated under reduced pressure.The residue was partitioned between water and dichloromethane, theaqueous layer extracted with dichloromethane, the organic layerscombined, dried (Na₂SO₄) and evaporated to a syrup. Flash silica gelcolumn chromatography using 2-3% gradient of methanol in ethyl acetateyielded 9 as a colorless foam (1.04 g, 63%). ¹H-NMR _(δ) 7.42 (d,J_(6,5)=8.4, 1H, H6 Urd), 7.53-6.97 (m, 13H, aromatic), 6.12 (d,J_(1,2′)=8.0, 1H, H-1′), 5.41 (m, 3H, H5 Urd, H4 NAcGal), 5.15 (dd,J_(3,4)=3.6, J_(3,2)=11.2, 2H, H₃NAcGal), 4.87 (dd, J_(2′,3′)=5.6,J_(2′,1′)=8.0, 1H, H2′), 4.63 (d, J_(1,2)=8.0, 2H, H1 NAcGal), 4.42 (d,J_(3′,2′)=5.6, 1H, H3′), 4.29-4.04 (m, 9H, H4′, H₂NAcGal, H₅NacGal,CH₂), 3.95-3.82 (m, 8H, H6 NAcGal, 2×OMe), 3.62-3.42 (m, 4H, H5′,H₆NAcGal), 3.26 (m, 2H, CH₂), 2.40-1.97 (m, 28H, CH₂, Ac), 1.95-1.30 (m,50H, CH₂). MS/ESI⁻ m/z 1727.0 (M−H)⁻.

2′-(N-α-bis-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose)-L-lysyl)amino-2′-deoxy-5′-O-4,4′-dimethoxytrityluridine 3′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (10).Conjugate 9 (0.87 g, 0.50 mmol) was dissolved in dry dichloromethane (10ml) under argon and diisopropylethylamine (0.36 ml, 2.07 mmol) and1-methylimidazole (21 μL, 0.26 mmol) were added. The solution was cooledto 0° C. and 2-cyanoethyl diisopropylchlorophosphoramidite (0.19 ml,0.85 mmol) was added. The reaction mixture was stirred at rt for 1 hour,than cooled to 0° C. and quenched with anhydrous ethanol (0.5 ml). Afterstirring for 10 min the solution was concentrated under reduced pressure(40° C.) and the residue dissolved in dichloromethane andchromatographed on the column of silica gel using hexanes-ethyl acetate1:1, followed by ethyl acetate and finally ethyl acetate-acetone 1:1 (1%triethylamine was added to solvents) to afford the phosphoramidite 10(680 mg, 69%). ³¹P-NMR _(δ) 152.0 (s), 149.3 (s). MS/ESI⁻ m/z 1928.0(M−H)⁻.

N-(12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol(11).12′-Hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galac-topyranose7 (850 mg, 1.56 mmol) was dissolved in DMF (5 ml) and to the solutionN-hydroxysuccinimide (215 mg, 1.87 mmol) and 1,3-dicyclohexylcarbodimide(386 mg, 1.87 mmol) were added. The reaction mixture was stirred at rtovernight, the precipitate was filtered off and to the filtrateD-threoninol (197 mg, 1.87 mmol) was added. The mixture was stirred atrt overnight and concentrated in vacuo. The residue was partitionedbetween dichloromethane and 5% NaHCO₃, the organic layer was washed withbrine, dried (Na₂SO₄) and evaporated to a syrup. Silica gel columnchromatography using 1-10% gradient of methanol in dichloromethaneafforded 11 as a colorless oil (0.7 g, 71%). ¹H-NMR δ6.35 (d, J=7.6, 1H,NH), 5.77 (d, J=8.0, 1H, NH), 5.44 (d, _(J4,3)=3.6, 1H, H4), 5.37 (dd,_(J3,4)=3.6, _(J3,2)=11.2, 1H, H3), 4.77 (d, _(J1,2)=8.0, 1H, H1),4.28-4.18 (m, 3H, CH₂, CH), 4.07-3.87 (m, 6H), 3.55 (m, 1H, H5), 3.09(d, J=3.2, 1H, OH), 3.02 (t, J=4.6, 1H, OH), 2.34 (t, J=7.4 2H, CH₂),2.23 (s, 3H, Ac), 2.10 (s, 3H, Ac), 2.04 (s, 3H, Ac), 1.76-1.61 (m,2×CH₂), 1.35 (m, 14H, 7×CH₂), 1.29 (d, J=6.4, 3H, CH₃). MS/ESI⁻ m/z(M−H)⁻.

1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol(12). To the solution of 11 (680 mg, 1.1 mmol) in dry pyridine (10 ml)p-anisylchlorotriphenylmethane (430 mg, 1.39 mmol) was added and thereaction mixture was stirred, protected from moisture, overnight.Methanol (3 ml) was added and the solution stirred for 15 min andevaporated in vacuo. The residue was partitioned between dichloromethaneand 5% NaHCO₃, the organic layer was washed with brine, dried (Na₂SO₄)and evaporated to a syrup. Silica gel column chromatography using 1-3%gradient of methanol in dichloromethane afforded 12 as a white foam(0.75 g, 77%). ¹H-NMR δ7.48-6.92 (m, 14H, aromatic), 6.15 (d, J=8.8, 1H,NH), 5.56 (d, J=8.0, 1H, NH), 5.45 (d, _(J4,3)=3.2, 1H, H4), 5.40 (dd,_(J3,4)=3.2, _(J3,2)=11.2, 1H, H3), 4.80 (d, _(J1,2)=8.0, 1H, H1),4.3-4.13 (m, 3H, CH₂, CH), 4.25-3.92 (m, 4H, H6, H2, CH), 3.89 (s, 3H,OMe), 3.54 (m, 2H, H5, CH), 3.36 (dd, J=3.4, J=9.8, 1H, CH), 3.12 (d,J=2.8, 1H, OH), 2.31 (t, J=7.6, 2H, CH₂), 2.22 (s, 3H, Ac), 2.13 (s, 3H,Ac), 2.03 (s, 3H, Ac), 1.80-1.55 (m, 2×CH₂), 1.37 (m, 14H, 7×CH₂), 1.21(d, J=6.4, 3H, CH₃). MS/ESI⁻ m/z 903.5 (M−H)⁻.

1-O-(4-Monomethoxytrityl)-N-(12′-hydroxydodecanoyl-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-galactopyranose)-D-threoninol3-O-(2-cyanoethyl N,N-diisopropylphosphorami-dite) (13). Conjugate 12(1.2 g, 1.33 mmol) was dissolved in dry dichloromethane (15 ml) underargon and diisopropylethylamine (0.94 ml, 5.40 mmol) and1-methylimidazole (55 μL, 0.69 mmol) were added. The solution was cooledto 0° C. and 2-cyanoethyl N,N-diisopropyl-chlorophosphoramidite (0.51ml, 2.29 mmol) was added. The reaction mixture was stirred at rt for 2hours, than cooled to 0° C. and quenched with anhydrous ethanol (0.5ml). After stirring for 10 min. the solution was concentrated underreduced pressure (40° C.) and the residue dissolved in dichloromethaneand chromatographed on the column of silica gel using 50-80% gradient ofethyl acetate in hexanes (1% triethylamine) to afford thephosphoramidite 13 (1.2 g, 82%). ³¹P-NMR

149.41 (s), 149.23 (s).

Oligonucleotide Synthesis

Phosphoramidites 10, and 13, were used along with standard 2′-O-TBDMSand 2′-O-methyl nucleoside phosphoramidites. Synthesis were conducted ona 394 (ABI) synthesizer using modified 2.5 μmol scale protocol with a 5min coupling step for 2′-O-TBDMS protected nucleotides and 2.5 mincoupling step for 2′-O-methyl nucleosides. Coupling efficiency for thephosphoramidite 10 was lower than 50% while coupling efficiencies forphosphoramidite 13 was typically greater than 95% based on themeasurement of released trityl cations. Once the synthesis wascompleted, the oligonucleotides were deprotected. The 5′-trityl groupswere left attached to the oligomers to assist purification. Cleavagefrom the solid support and the removal of the protecting groups wasperformed as described herein with the exception of using 20% piperidinein DMF for 15 min for the removal of Fm protection prior methylaminetreatment. The 5′-tritylated oligomers were separated from shorter(trityl-off) failure sequences using a short column of SEP-PAK C-18adsorbent. The bound, tritylated oligomers were detritylated on thecolumn by treatment with 1% trifluoroacetic acid, neutralized withtriethylammonium acetate buffer, and than eluted. Further purificationwas achieved by reverse-phase HPLC. An example of aN-acetyl-D-galactosamine conjugate that can be synthesized usingphosphoramidite 13 is shown in FIG. 15.

Structures of the ribozyme conjugates were confirmed by MALDI-TOF MS.

Monomer Synthesis

2′-Amino-2′-deoxyuridine-N-acetyl-D-galactosamine conjugate. Thebis-Fmoc protected lysine linker was attached to the 2′-amino group of2′-amino-2′-deoxyuridine using the EEDQ catalyzed peptide coupling. The5′-OH was protected with 4,4′-dimethoxytrityl group to give 1, followedby the cleavage of N-Fmoc groups with diethylamine to afford synthon 2in the high overall yield.

2-acetamido-3,4,6-tetra-O-acetyl-1-chloro-D-galactopyranose 4 wassynthesized with minor modifications according to the reported procedure(Findeis supra). Mercury salt catalyzed glycosylation of 4 with thebenzyl ester of 12-hydroxydodecanoic acid 5 afforded glycoside 6 in 81%yield. Hydrogenolysis of benzyl protecting group yielded 7 in aquantitative yield. The coupling of the sugar derivative with thenucleoside synthon was achieved through preactivation of the carboxylicfunction of 7 as N-hydroxysuccinimide ester 8, followed by coupling tolysyl-2′-aminouridine conjugate 2. The final conjugate 9 was thanphosphitylated under standard conditions to afford the phosphoramidite10 in 69% yield.

D-Threoninol-N-acetyl-D-galactosamine conjugate Using the similarstrategy as described above, D-threoninol was coupled to 7 to affordconjugate 11 in a good yield. Monomethoxytritylation, followed byphosphitylation yielded the desired phosphoramidite 13.

Synthesis of Oxime Linked Nucleic Acid/Peptide Conjugates (FIGS. 16 and17)

12-Hydroxydodecanoic acid benzyl ester Benzyl bromide (10.28 ml, 86.45mmol) was added dropwise to a solution of 12-hydroxydodecanoic acid (17g, 78.59 mmol) and DBU ml, 86.45 mmol) in absolute DMF (120 ml) undervigorous stirring at 0° C. After completion of the addition reactionmixture was warmed to a room temperature and left overnight understirring. TLC (hexane-ethylacetate 3:1) indicated completetransformation of the starting material. DMF was removed under reducedpressure and the residue was partitioned between ethyl ether and 1N HCl.Organic phase was separated, washed with saturated aq sodium bicarbonateand dried over sodium sulfate. Sodium sulfate was filtered off, filtratewas evaporated to dryness. The residue was crystallized from hexane togive 21.15 g (92%) of the title compound as a white powder.

12-O—N-Phthaloyl-dodecanoic acid benzyl ester (15).Diethylazodicarboxylate (DEAD, 16.96 ml, 107.7 mmol) was added dropwiseto the mixture of 12-Hydroxydodecanoic acid benzyl ester (21 g, 71.8mmol), triphenylphosphine (28.29 g, 107.7 mmol) and N-hydroxyphthalimide(12.88 g, 78.98 mmol) in absolute THF (250 ml) at −20°-−30° C. understirring. The reaction mixture was stirred at this temperature foradditional 2-3 h, after which time TLC (hexane-ethylacetate 3:1)indicated reaction completion. The solvent was removed in vacuo and theresidue was treated ether (250 ml). Formed precipitate oftriphenylphosphine oxide was filtered off, mother liquor was evaporatedto dryness and the residue was dissolved in methylene chloride andpurified by flash chromatography on silica gel in hexane-ethyl acetate(7:3). Appropriate fractions were pooled and evaporated to dryness toafford 26.5 g (84.4%) of compound 15.

12-O—N-Phthaloyl-dodecanoic acid (16). Compound 15 (26.2 g, 59.9 mmol)was dissolved in 225 ml of ethanol-ethylacetate (3.5:1) mixture and 10%Pd/C (2.6 g) was added. The reaction mixture was hydrogenated in Parrapparatus for 3 hours. Reaction mixture was filtered through celite andevaporated to dryness. The residue was crystallized from methanol toprovide 15.64 g (75%) of compound 16.

12-O—N-Phthaloyl-dodecanoic acid 2,3-di-hydroxy-propylamide (18) Themixture of compound 16 (15.03 g, 44.04 mmol), dicyclohexylcarbodiimide(10.9 g, 52.85 mmol) and N-hydroxysuccinimide (6.08 g, 52.85 mmol) inabsolute DMF (150 ml) was stirred at room temperature overnight. TLC(methylene chloride-methanol 9:1) indicated complete conversion of thestarting material and formation of NHS ester 17. Then aminopropanediol(4.01 g, 44 mmol) was added and the reaction mixture was stirred at roomtemperature for another 2 h. The formed precipitate of dicyclohexylureawas removed by filtration, filtrate was evaporated under reducedpressure. The residue was partitioned between ethyl acetate andsaturated aq sodium bicarbonate. The whole mixture was filtered toremove any insoluble material and clear layers were separated. Organicphase was concentrated in vacuo until formation of crystalline material.The precipitate was filtered off and washed with cold ethylacetate toproduce 10.86 g of compound 17. Combined mother liquor and washings wereevaporated to dryness and crystallized from ethylacetate to afford 3.21g of compound 18. Combined yield—14.07 g (73.5%).

12-O—N-Phthaloyl-dodecanoic acid2-hydroxy,3-dimethoxytrityloxy-propylamide (19) Dimethoxytrityl chloride(12.07 g, 35.62 mmol) was added to a stirred solution of compound 18(14.07 g, 32.38 mmol) in absolute pyridine (130 ml) at 0° C. Thereaction solution was kept at 0° C. overnight. Then it was quenched withMeOH (10 ml) and evaporated to dryness. The residue was dissolved inmethylene chloride and washed with saturated aq sodium bicarbonate.Organic phase was separated, dried over sodium sulfate and evaporated todryness. The residue was purified by flash chromatography on silica gelusing step gradient of acetone in hexanes (3:7 to 1:1) as an eluent.Appropriate fractions were pooled and evaporated to provide 14.73 g(62%) of compound 19, as a colorless oil.

12-O—N-Phthaloyl-dodecanoic acid2-O-(cyanoethyl-N,N-diisopropylamino-phosphoramidite),3-dimethoxytrityloxy-propylamide(20). Phosphitylated according to Sanghvi, et al., 2000, Organic ProcessResearch and Development, 4, 175-81.

Purified by flash chromatography on silica gel using step gradient ofacetone in hexanes (1:4 to 3:7) containing 0.5% of triethylamine.Yield—82%, colourless oil.

Oxidation of Peptides

Peptide (3.3 mg, 3.3 μmol) was dissolved in 10 mM AcONa and 2 eq ofsodium periodate (100 mM soln in water) was added. Final reactionvolume—0.5 ml. After 10 minutes reaction mixture was purified usinganalytical HPLC on Phenomenex Jupiter 5u C18 300A (150×4.6 mm) column;solvent A: 50 mM KH₂PO₄ (pH 3); solvent B: 30% of solvent A in MeCN;gradient B over 30 min. Appropriate fractions were pooled andconcentrated on a SpeedVac to dryness. Yield: quantitative.

Conjugation Reaction of Herzyme-ONH2-1Lnker with N-Glyoxyl Peptide (FIG.17)

Herzyme (SEQ ID NO: 13) with a 5′-terminal linker (100 OD) was mixedwith oxidized peptide (3-5 eq) in 50 mM KH2PO4 (pH3, reaction volume 1ml) and kept at room temperature for 24-48 h. The reaction mixture waspurified using analytical HPLC on a Phenomenex Jupiter 5u C18 300A(150×4.6 mm) column; solvent A: 10 mM TEAA; solvent B: 10 mM TEAA/MeCN.Appropriate fractions were pooled and concentrated on a SpeedVac todryness to provide desired conjugate. ESMS: calculated: 12699,determined: 12698.

Example 5 Synthesis of Phospholipid Enzymatic Nucleic Acid Conjugates(FIG. 19)

A phospholipid enzymatic nucleic acid conjugate (see FIG. 19) wasprepared by coupling a C18H37 phosphoramidite to the 5′-end of anenzymatic nucleic acid molecule (Angiozyme™, SEQ ID NO: 24) during solidphase oligonucleotide synthesis on an ABI 394 synthesizer using standardsynthesis chemistry. A 5′-terminal linker comprising3′-AdT-di-Glycerol-5′, where A is Adenosine, dT is 2′-deoxy Thymidine,and di-Glycerol is a di-DMT-Glycerol linker (Chemgenes CAT numberCLP-5215), is used to attach two C18H37 phosphoramidites to theenzymatic nucleic acid molecule using standard synthesis chemistry.Additional equivalents of the C18H37 phosphoramidite were used for thebis-coupling. Similarly, other nucleic acid conjugates as shown in FIG.18 can be prepared according to similar methodology.

Example 6 Synthesis of Peg Enzymatic Nucleic Acid Conjugates (FIG. 20)

A 40K-PEG enzymatic nucleic acid conjugate (see FIG. 20) was prepared bypost synthetic N-hydroxysuccinimide ester coupling of a PEG derivative(Shearwater Polymers Inc, CAT number PEG2-NHS) to the 5′-end of anenzymatic nucleic acid molecule (Angiozyme™, SEQ ID NO: 24). A5′-terminal linker comprising 3′-AdT-C6-amine-5′, where A is Adenosine,dT-C6-amine is 2′-deoxy Thymidine with a C5 linked six carbon aminelinker (Glen Research CAT number 10-1039-05), is used to attach the PEGderivative to the enzymatic nucleic acid molecule using NHS couplingchemistry.

Angiozyme™ with the C6dT-NH2 at the 5′ end was synthesized anddeprotected using standard oligonucleotide synthesis procedures asdescribed herein. The crude sample was subsequently loaded onto areverse phase column and rinsed with sodium chloride solution (0.5 M).The sample was then desalted with water on the column until theconcentration of sodium chloride was close to zero. Acetonitrile wasused to elute the sample from the column. The crude product was thenconcentrated and lyophilized to dryness.

The crude material (Angiozyme™) with 5′-amino linker (50 mg) wasdissolved in sodium borate buffer (1.0 mL, pH 9.0). The PEG NHS ester(200 mg) was dissolved in anhydrous DMF (1.0 mL). The Angiozyme™ buffersolution was then added to the PEG NHS ester solution. The mixture wasimmediately vortexed for 5 minutes. Sodium acetate buffer solution (5mL, pH 5.2) was used to quench the reaction. Conjugated material wasthen purified by ion-exchange and reverse phase chromatography.

Example 7 Phamacokinetics of PEG ribozyme acid conjugate (FIG. 21)

Forty-eight female C57B1/6 mice were given a single subcutaneous (SC)bolus of 30 mg/kg Angiozyme™ and 30 mg/kg Angiozyme™/40K PEG conjugate.Plasma was collected out to 24 hours post ribozyme injection. Plasmasamples were analyzed for full length ribozyme by a hybridization assay.

Oligonucleotides complimentary to the 5′ and 3′ ends of Angiozyme™ weresynthesized with biotin at one oligo, and FITC on the other oligo. Abiotin oligo and FITC labeled oligo pair are incubated at 1 ug/ml withknown concentrations of Angiozyme™ at 75 degrees C. for 5 min. After 10minutes at RT, the mixture is allowed to bind to streptavidin coatedwells of a 96-wll plate for two hours. The plate is washed withTris-saline and detergent, and peroxidase labeled anti-FITC antibody isadded. After one hour, the wells are washed, and the enzymatic reactionis developed, then read on an ELISA plate reader. Results are shown inFIG. 21.

Example 8 Phamacokinetics of Phospholipid Ribozyme Conjugate (FIG. 22)

Seventy-two female C57B1/6 mice were given a single intravenous (4)bolus of 30 mg/kg Angiozyme™ and 30 mg/kg Angiozyme™ conjugated withphospholipid (FIG. 19). Plasma was collected out to 3 hours postribozyme injection. Plasma samples were analyzed for full lengthribozyme by a hybridization assay.

Oligonucleotides complimentary to the 5′ and 3′ ends of Angiozyme™ weresynthesized with biotin at one oligo, and FITC on the other oligo. Abiotin oligo and FITC labeled oligo pair are incubated at 1 ug/ml withknown concentrations of Angiozyme™ at 75 degrees C. for 5 min. After 10minutes at RT, the mixture is allowed to bind to streptavidin coatedwells of a 96-wll plate for two hours. The plate is washed withTris-saline and detergent, and peroxidase labeled anti-FITC antibody isadded. After one hr, the wells are washed, and the enzymatic reaction isdeveloped, then read on an ELISA plate reader. Results are shown in FIG.22.

Example 9 Synthesis of Protein or Peptide Conjugates with BiodegradableLinkers (FIGS. 24-26, and 29)

Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using oxime and morpholino linkages. For example, atherapeutic antibody can be conjugated with PEG to improve the FIG. 24shows a non-limiting example of a synthetic approach for synthesizingpeptide or protein conjugates to PEG utilizing a biodegradable linker,the example shown is for a protein conjugate. Other conjugates can besynthesized in a similar manner where the protein or peptide isconjugated to molecules other than PEG, such as small molecules, toxins,radioisotopes, peptides or other proteins. (a) The protein of interest,such as an antibody or interferon, is synthesized with a terminal Serineor Threonine moiety that is oxidized, for example with sodium periodate.The oxidized protein is then coupled to a nucleic acid linker moleculethat is designed to be biodegradable, for example acytidine-deoxythymidine, cytidine-deoxyuridine,adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that containsan oxyamino (O—NH₂) function. Other biodegradable nucleic acid linkerscan be similarly used, for example other dimers, trimers, tetramers etc.that are designed to be biodegradable. The example shown makes use of a5′-oxyamino moiety, however, other examples can utilize an oxyamino atother positions within the nucleic acid molecule, for example at the2′-position, 3′-position, or at a nucleic acid base position. (b) Theprotein/nucleic acid conjugate is then oxidized to generate a dialdehydefunction that is coupled to PEG molecule comprising an amino group(H₂N-PEG), for example a PEG molecule with an amino linker. Other aminocontaining molecules can be conjugated as shown in the figure, forexample small molecules, toxins, or radioisotope labeled molecules.

Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using oxime and phosphoramidate linkages. FIG. 25 shows anon-limiting example of a synthetic approach for synthesizing peptide orprotein conjugates to PEG utilizing a biodegradable linker, the exampleshown is for a protein conjugate. Other conjugates can be synthesized ina similar manner where the protein or peptide is conjugated to moleculesother than PEG, such as small molecules, toxins, radioisotopes, peptidesor other proteins. The protein of interest, such as an antibody orinterferon, is synthesized with a terminal Serine or Threonine moietythat is oxidized, for example with sodium periodate. The oxidizedprotein is then coupled to a nucleic acid linker molecule that isdesigned to be biodegradable, for example a cytidine-deoxythymidine,cytidine-deoxyuridine, adenosine-deoxythymidine, oradenosine-deoxyuridine dimer that contains an oxyamino (O—NH₂) functionand a terminal phosphate group. Terminal phosphate groups can beintroduced during synthesis of the nucleic acid molecule using chemicalphosphorylation reagents, such as Glen Research Cat Nos. 10-1909-02,10-1913-02, 10-1914-02, and 10-1918-02. Other biodegradable nucleic acidlinkers can be similarly used, for example other dimers, trimers,tetramers etc. that are designed to be biodegradable. The example shownmakes use of a 5′-oxyamino moiety, however, other examples can utilizean oxyamino at other positions within the nucleic acid molecule, forexample at the 2′-position, 3′-position, or at a nucleic acid baseposition. The protein/nucleic acid conjugate terminal phosphate group isthen activated with an activator reagent, such as NMI and/or tetrazole,and coupled a PEG molecule comprising an amino group (H₂N-PEG), forexample a PEG molecule with an amino linker. Other amino containingmolecules can be conjugated as shown in the figure, for example smallmolecules, toxins, or radioisotope labeled molecules.

Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using phosphoramidate linkages. FIG. 26 shows a non-limitingexample of a synthetic approach for synthesizing peptide or proteinconjugates to PEG utilizing a biodegradable linker, the example shown isfor a protein conjugate. Other conjugates can be synthesized in asimilar manner where the protein or peptide is conjugated to moleculesother than PEG, such as small molecules, toxins, radioisotopes, peptidesor other proteins. (a) A nucleic acid linker molecule that is designedto be biodegradable, for example a cytidine-deoxythymidine,cytidine-deoxyuridine, adenosine-deoxythymidine, oradenosine-deoxyuridine dimer, is synthesized with a terminal phosphategroup. Other biodegradable nucleic acid linkers can be similarly used,for example other dimers, trimers, tetramers etc. that are designed tobe biodegradable. The protein/nucleic acid conjugate terminal phosphategroup is then activated with an activator reagent, such as NMI and/ortetrazole, and coupled a PEG molecule comprising an amino group(H₂N-PEG), for example a PEG molecule with an amino linker. Other aminocontaining molecules can be conjugated as shown in the figure, forexample small molecules, toxins, or radioisotope labeled molecules. Theterminal protecting group, for example a dimethoxytrityl group, isremoved from the conjugate and a terminal phosphite group is introducedwith a phosphitylating reagent, such as N,N-diisopropyl-2-cyanoethylchlorophosphoramidite. (b) The PEG/nucleic acid conjugate is thencoupled to a peptide or protein comprising an amino group, such as theamino terminus or amino side chain of a suitably protected peptide orprotein or via an amino linker. The conjugate is then oxidized and anyprotecting groups are removed to yield the protein/PEG conjugatecomprising a biodegradable linker.

Proteins and peptides can be conjugated with various molecules,including PEG, via biodegradable nucleic acid linker molecules of theinvention, using phosphoramidate linkages from coupling protein-basedphosphoramidites. FIG. 29 shows a non-limiting example of a syntheticapproach for synthesizing peptide or protein conjugates to PEG utilizinga biodegradable linker, the example shown is for a protein conjugate.Other conjugates can be synthesized in a similar manner where theprotein or peptide is conjugated to molecules other than PEG, such assmall molecules, toxins, radioisotopes, peptides or other proteins. Theprotein of interest, such as an antibody or interferon, is synthesizedwith a terminal Serine, Threonin, or Tyrosine moiety that isphosphitylated, for example with N,N-diisopropyl-2-cyanoethylchlorophosphoramidite. The phosphitylated protein is then coupled to anucleic acid linker molecule that is designed to be biodegradable, forexample a cytidine-deoxythymidine, cytidine-deoxyuridine,adenosine-deoxythymidine, or adenosine-deoxyuridine dimer that containsconjugated PEG molecule as described in FIG. 18. Other biodegradablenucleic acid linkers can be similarly used, for example other dimers,trimers, tetramers etc. that are designed to be biodegradable.

Example 10 Galactosamine Ribozyme Conjugate Targeting HBV

A nuclease-resistance ribozyme directed against the Hepatitis B viralRNA (HBV) (HepBzyme™) is in early stages of preclinical development.HepBzyme, which targets site 273 of the Hepatitis B viral RNA, hasproduced statistically significant decreases in serum HBV levels in aHBV transgenic mouse model in a dose-dependent manner (30 and 100mg/kg/day). In an effort to improve hepatic uptake by targeting theasialoglycoprotein receptor, a series of 5 branched galactosamineresidues were attached via phosphate linkages to the 5′-terminus ofHepBzyme (Gal-HepBzyme). The affect of the galactosamine conjugation onHepBzyme was assessed by quantitation of ³²P-labeled HepBzyme andGal-HepBzyme in plasma, liver and kidney of mice following a single SCbolus administration of 30 mg/kg. The plasma disposition of the intactribozyme was similar between Gal-HepBzyme and HepBzyme. An approximatethree-fold increase in the maximum observed concentration of intactribozyme in liver (C_(max)) was observed in liver for Gal-HepBzyme(6.1±1.8 ng/mg) vs. HepBzyme (2.2±0.8 ng/mg) (p<0.05). The area underthe curve (AUCall) for Gal-HepBzyme was also increased by approximatelytwo-fold. This was accompanied by a substantial decrease (approximately40%) in the AUCall for intact ribozyme in kidney. In addition to thesignificant increase in C_(max) observed for intact Gal-HepBzyme in theliver, there was an increase in the total number of ribozymeequivalents, which may be suggestive of increased affinity of both theintact ribozyme and metabolites for asialoglycoprotein receptor andgalactose-specific receptors in the liver. These data demonstrate thatconjugation of a ribozyme with galactosamine produces a compound with amore favorable disposition profile, and illustrates the utility ofconjugated ribozymes with improved in vivo pharmacokinetics andbiodistribution.

Example 11 Synthesis of siNA Conjugates

siNA molecules can be designed to interact with various sites in atarget RNA message, for example, target sequences within the RNAsequence. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences. The siNA molecules can bechemically synthesized using methods described herein. Inactive siNAmolecules that are used as control sequences can be synthesized byscrambling the sequence of the siNA molecules such that it is notcomplementary to the target sequence. Generally, siNA constructs can bysynthesized using solid phase oligonucleotide synthesis methods asdescribed herein (see for example Usman et al., U.S. Pat. Nos.5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098; 6,362,323;6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos. 6,111,086;6,008,400; 6,111,086 all incorporated by reference herein in theirentirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Deprotection and purification of the siNA can beperformed as is generally described in Usman et al., U.S. Pat. No.5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellonet al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No.6,303,773, or Scaringe supra, incorporated by reference herein in theirentireties. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of siNA constructs. Forexample, applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

The introduction of conjugate moieties is accomplished either duringsolid phase synthesis using phosphoramidite chemistry described above,or post-synthetically using, for example, N-hydroxysuccinimide (NHS)ester coupling to an amino linker present in the siNA. Typically, aconjugate introduced during solid phase synthesis will be added to the5′-end of a nucleic acid sequence as the final coupling reaction in thesynthesis cycle using the phosphoramidite approach. Coupling conditionscan be optimized for high yield coupling, for example by modification ofcoupling times and reagent concentrations to effectuate efficientcoupling. As such, the 5′-end of the sense strand of a siNA molecule isreadily conjugated with a conjugate moiety having a reactive phosphorusgroup available for coupling (e.g., a compound having Formulae 1, 5, 8,55, 56, 57, 60, 86, 92, 104, 110, 113, 115, 116, 117, 118, 120, or 122)using the phosphoramidite approach, providing a 5′-terminal conjugate(see for example FIG. 41).

Conjugate precursors having a reactive phosphorus group and a protectedhydroxyl group can be used to incorporate a conjugate moiety anywhere inthe siNA sequence, such as in the loop portion of a single strandedhairpin siNA construct (see for example FIG. 42). For example, using thephosphoramidite approach, a conjugate moiety comprising aphosphoramidite and protected hydroxyl (e.g., a compound having Formulae86, 92, 104, 113, 115, 116, 117, 118, 120, or 122 herein) is firstcoupled at the desired position within the siNA sequence using solidphase synthesis phosphoramidite coupling. Second, removal of theprotecting group (e.g., dimethoxytrityl) allows coupling of additionalnucleotides to the siNA sequence. This approach allows the conjugatemoiety to be positioned anywhere within the siNA molecule.

Conjugate derivatives can also be introduced to a siNA molecule postsynthetically. Post synthetic conjugation allows a conjugate moiety tobe introduced at any position within the siNA molecule where anappropriate functional group is present (e.g., a C5 alkylamine linkerpresent on a nucleotide base or a 2′-alkylamine linker present on anucleotide sugar can provide a point of attachment for an NHS-conjugatemoiety). Generally, a reactive chemical group present in the siNAmolecule is unmasked following synthesis, thus allowing post-syntheticcoupling of the conjugate to occur. In a non-limiting example, anprotected amino linker containing nucleotide (e.g., TFA protected C5propylamino thymidine) is introduced at a desired position of the siNAduring solid phase synthesis. Following cleavage and deprotection of thesiNA, the free amine is made available for NHS ester coupling of theconjugate at the desired position within the siNA sequence, such as atthe 3′-end of the sense and/or antisense strands, the 3′ and/or 5′-endof the sense strand, or within the siNA sequence, such as in the loopportion of a single stranded hairpin siNA sequence.

A conjugate moiety can be introduced at different locations within asiNA molecule using both solid phase synthesis and post-syntheticcoupling approaches. For example, solid phase synthesis can be used tointroduce a conjugate moiety at the 5′-end of the siNA (e.g. sensestrand) and post-synthetic coupling can be used to introduce a conjugatemoiety at the 3′-end of the siNA (e.g. sense strand and/or antisensestrand). As such, a siNA sense strand having 3′ and 5′ end conjugatescan be synthesized (see for example FIG. 41). Conjugate moieties canalso be introduced in other combinations, such as at the 5′-end, 3′-endand/or loop portions of a siNA molecule (see for example FIG. 42).

Example 12 Phamacokinetics of siNA Conjugates (FIG. 43)

Three nuclease resistant siNA molecule targeting site 1580 of hepatitisB virus (HBV) RNA were designed using Stab 7/8 chemistry (see Table IV)and a 5′-terminal conjugate moiety.

One siNA conjugate comprises a branched cholesterol conjugate linked tothe sense strand of the siNA. The “cholesterol” siNA conjugate moleculehas the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

Another siNA conjugate comprises a branched phospholipid conjugatelinked to the sense strand of the siNA. The “phospholipid” siNAconjugate molecule has the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

Another siNA conjugate comprises a polyethylene glycol (PEG) conjugatelinked to the sense strand of the siNA. The “PEG” siNA conjugatemolecule has the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

The Cholesterol, Phospholipid, and PEG conjugates were evaluated forpharmakokinetic properties in mice compared to a non-conjugated siNAconstruct having matched chemistry and sequence. This study wasconducted in female CD-1 mice approximately 26 g (6-7 weeks of age).Animals were housed in groups of 3. Food and water were provided adlibitum. Temperature and humidity were according to Pharmacology TestingFacility performance standards (SOP's) which are in accordance with the1996 Guide for the Care and Use of Laboratory Animals (NRC). Animalswere acclimated to the facility for at least 3 days prior toexperimentation.

Absorbance at 260 nm was used to determine the actual concentration ofthe stock solution of pre-annealed HBV siNA. An appropriate amount ofHBV siNA was diluted in sterile veterinary grade normal saline (0.9%)based on the average body weight of the mice. A small amount of theantisense (Stab 7) strand was internally labeled with gamma 32P-ATP. The32P-labeled stock was combined with excess sense strand (Stab 8) andannealed. Annealing was confirmed prior to combination with unlableddrug. Each mouse received a subcutaneous bolus of 30 mg/kg (based onduplex) and approximately 10 million cpm (specific activity ofapproximately 15 cpm/ng).

Three animals per timepoint (1, 4, 8, 24, 72, 96 h) were euthanized byCO₂ inhalation followed immediately by exsanguination. Blood was sampledfrom the heart and collected in heparinized tubes. After exsanguination,animals were perfused with 10-15 mL of sterile veterinary grade salinevia the heart. Samples of liver were then collected and frozen.

Tissue samples were homogenized in a digestion buffer prior to compoundquantitation. Quantitation of intact compound was determined byscintillation counting followed by PAGE and phosphorimage analysis.Results are shown in FIG. 43. As shown in the figure, the conjugatedsiNA constructs shown vastly improved liver PK compared to theunconjugated siNA construct.

Example 13 Cell Culture of siNA Conjugates (FIG. 44)

The Cholesterol conjugates and Phospholipid conjugated siNA constructsdescribed in Example 12 above were evaluated for cell culture efficacyin a HBV cell culture system.

Transfection of HepG2 Cells with psHBV-1 and siNA

The human hepatocellular carcinoma cell line Hep G2 was grown inDulbecco's modified Eagle media supplemented with 10% fetal calf serum,2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. Togenerate a replication competent cDNA, prior to transfection the HBVgenomic sequences are excised from the bacterial plasmid sequencecontained in the psHBV-1 vector. Other methods known in the art can beused to generate a replication competent cDNA. This was done with anEcoRI and Hind III restriction digest. Following completion of thedigest, a ligation was performed under dilute conditions (20 μg/ml) tofavor intermolecular ligation. The total ligation mixture was thenconcentrated using Qiagen spin columns.

siNA Activity Screen and Dose Response Assay

Transfection of the human hepatocellular carcinoma cell line, Hep G2,with replication-competent HBV DNA results in the expression of HBVproteins and the production of virions. To test the efficacy of siNAconjugates targeted against HBV RNA, the Cholesterol siNA conjugate andPhospholipid siNA conjugate described in Example 12 were compared to anon-conjugated control siNA (see FIG. 44). An inverted sequence duplexwas used as a negative control for the unconjugated siNA. Dose responsestudies were performed in which HBV genomic DNA was transfected with HBVgenomic DNA with lipid at 12.5 ug/ml into Hep G2 cells. 24 hours aftertransfection with HBV DNA, cell culture media was removed and siNAduplexes were added to cells without lipid at 10 uM, 5, uM, 2.5 uM, 1uM, and 100 nm and the subsequent levels of secreted HBV surface antigen(HBsAg) were analyzed by ELISA 72 hours post treatment (see FIG. 44). Todetermine siNA activity, HbsAg levels were measured followingtransfection with siNA. Immulon 4 (Dynax) microtiter wells were coatedovernight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1μg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO3 35 mM, pH 9.5). Thewells were then washed 4× with PBST (PBS, 0.05% Tween® 20) and blockedfor 1 hr at 37° C. with PBST, 1% BSA. Following washing as above, thewells were dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg(Accurate YVS1807) was diluted 1:1000 in PBST and incubated in the wellsfor 1 hr. at 37° C. The wells were washed 4× with PBST.Streptavidin/Alkaline Phosphatase Conjugate (Pierce 21324) was dilutedto 250 ng/ml in PBST, and incubated in the wells for 1 hr. at 37° C.After washing as above, p-nitrophenyl phosphate substrate (Pierce 37620)was added to the wells, which were then incubated for 1 hour at 37° C.The optical density at 405 nm was then determined. As shown in FIG. 44,the phospholipid and cholesterol conjugates demonstrate marked dosedependent inhibition of HBsAg expression compared to the unconjugatedsiNA construct when delivered to cells without any transfection agent(lipid).

Example 14 Purification of Nucleic Acid Conjugates

Nucleic acid conjugates of the invention can be purified using, forexample, anion exchange, reverse phase, and/or hydrophobic interactionchromoatography. Non-lipophilic nucleic acid conjugates of the invention(e.g., PEG, PEI, polyamine conjugates) are readily purified using anionexchange chromatography as is known in the art. Lipophilic nucleic acidconjugates of the invention (e.g., cholesterol, phospholipid, or alkylpolymer conjugates) can be purified using reverse phase chromatographyas is known in the art and/or by hydrophobic interaction chromatography.Hydrophobic interaction chromotography (HIC) allows high efficiencypurification of nucleic acid conjugates of the invention without the useof organic solvents.

Hydrophobic interaction chromatography is based on interactions betweenhydrophobic groups on the molecules to be purified or isolated and thecorresponding HIC resin. HIC utilizes such hydrophobic interactions inhighly polar non-denaturing buffers. Several different HIC resins can beutilized in purifying hydrophobic polynucleotide conjugates (e.g., siNAconjugates). Examples of HIC resins include but are not limited toether, phenyl, butyl, or hexyl stationary phases such as Toyopearl.650 SPhenyl, TSK GEL Phenyl-5PW, TSK GEL Ether-5PW, Toyopearl Ether-650,Toyopearl Phenyl-650, Toyopearl Butyl-650, and Toyopearl Hexyl-650 fromTosoHaas and Fractogel EMD Phenyl S from Merck. Various conditions thatcan be altered to achieve highly purified material using HIC includealterations in the concentration of salts in buffers, use of differentresins, varying pH and temperature.

In a non-limiting example, HIC was used to purify a Stab 7/8 (Table IV)siNA cholesterol conjugate having SEQ ID NO: 24 (see for example FIG.30) and a Stab 7/8 (Table IV) siNA phospholipid conjugate having SEQ IDNO: 26 (see for example FIG. 19). The purification buffer reagents usedin the HIC purification consisted of Ammonium Sulfate, Sodium PhosphateMonobasic and Dibasic (see Table V) which were purchased from VWR. Thematerial was purified on a Waters LC-2000 preparative system includingan LC Controller and pump and a waters 24487 dual wavelength UVDetector. The system is controlled by Millennium software version 4. Thebuffers and loading material is passed through a heat exchanger, such asa Timberline TL50D (Timberline Instruments (Boulder, Colo.). The Columnsused in the development included a Pharmacia HR 5/5, Pharmacia HR 16/10and a Pharmacia Fineline 35. The columns were packed with ToyopearlPhenyl-650s (Tosoh Bioscience, LLC Montgomeryville, Pa.) to a bed heightof 5 cm, 10 cm and 10 cm respectively as the process was scaled up.Further work included the Toyopearl 650 S Phenyl to the Tosoh BioscienceTSK GEL Phenyl-5PW and the Fractogel EMD Phenyl S.

The deprotected cholesterol siNA and phospholipid siNA conjugates werediluted in Milli-Q-water and ammonium sulfate was added to a 2Mconcentration following filtration and rinsing of the filter withMilli-Q-water. The addition of solid ammonium sulfate as a dry powderresulted in precipitation of the siNA conjugates. This process has beenfurther optimized so that the oligonucleotide is diluted inMilli-Q-water and then the volume is doubled with 2 M ammonium sulfateyielding a solution of 1 M ammonium sulfate with 10 mM Sodium Phosphate.

The purified conjugated siRNA is eluted from the column during step 2 ofthe gradient (see Table VI). This step also desalts the molecule. Theeluate was collected as fractions, which were analyzed by SAX or RP HPLCto determine purity. Fractions containing the conjugated product werepooled and this pool was analyzed by SAX or RP HPLC for purity. Thepooled material was stored 2-8° C. until annealed to the complementarystrand and desalted.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein are exemplary and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art, which are encompassedwithin the spirit of the invention, are defined by the scope of theclaims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed byvarious embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Other embodiments are within the following claims.

TABLE I Characteristics of naturally occurring ribozymes Group I IntronsSize: ~150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintenance of the active structure. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies [i, ii].Complete kinetic framework established for one ribozyme [iii, iv, v,vi]. Studies of ribozyme folding and substrate docking underway [vii,viii, ix]. Chemical modification investigation of important residueswell established [xxi]. The small (4-6 nt) binding site may make thisribozyme too non-specific for targeted RNA cleavage, however, theTetrahymena group I intron has been used to repair a “defective”β-galactosidase message by the ligation of new β-galactosidase sequencesonto the defective message [xii]. RNAse P RNA (M1 RNA) Size: ~290 to 400nucleotides. RNA portion of a ubiquitous ribonucleoprotein enzyme.Cleaves tRNA precursors to form mature tRNA [xiii]. Reaction mechanism:possible attack by M²⁺-OH to generate cleavage products with 3′-OH and5′-phosphate. RNAse P is found throughout the prokaryotes andeukaryotes. The RNA subunit has been sequenced from bacteria, yeast,rodents, and primates. Recruitment of endogenous RNAse P for therapeuticapplications is possible through hybridization of an External GuideSequence (EGS) to the target RNA [xiv, xv] Important phosphate and 2′ OHcontacts recently identified [xvi, xvii] Group II Introns Size: >1000nucleotides. Trans cleavage of target RNAs recently demonstrated [xviii,xvix]. Sequence requirements not fully determined. Reaction mechanism:2′-OH of an internal adenosine generates cleavage products with 3′-OHand a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Onlynatural ribozyme with demonstrated participation in DNA cleavage [xx,xxi] in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [xxii]. Important2′ OH contacts beginning to be identified [xxiii] Kinetic frameworkunder development [xxiv] Neurospora VS RNA Size: ~144 nucleotides. Transcleavage of hairpin target RNAs recently demonstrated [xxv]. Sequencerequirements not fully determined. Reaction mechanism: attack by 2′-OH5′ to the scissile bond to generate cleavage products with 2′,3′-cyclicphosphate and 5′-OH ends. Binding sites and structural requirements notfully determined. Only 1 known member of this class. Found in NeurosporaVS RNA. Hammerhead Ribozyme (see text for references) Size: ~13 to 40nucleotides. Requires the target sequence UH immediately 5′ of thecleavage site. Binds a variable number nucleotides on both sides of thecleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. 14 known members of this class. Found in a number of plantpathogens (virusoids) that use RNA as the infectious agent. Essentialstructural features largely defined, including 2 crystal structures[xxvi, xxvii] Minimal ligation activity demonstrated (for engineeringthrough in vitro selection) [xxviii] Complete kinetic frameworkestablished for two or more ribozymes [xxix]. Chemical modificationinvestigation of important residues well established [xxx]. HairpinRibozyme Size: ~50 nucleotides. Requires the target sequence GUCimmediately 3′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site and a variable number to the 3′-side of thecleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. 3 known members of this class. Found in three plant pathogen(satellite RNAs of the tobacco ringspot virus, arabis mosaic virus andchicory yellow mottle virus) which uses RNA as the infectious agent.Essential structural features largely defined [xxxi, xxxii, xxxiii,xxxiv] Ligation activity (in addition to cleavage activity) makesribozyme amenable to engineering through in vitro selection [xxxv]Complete kinetic framework established for one ribozyme [xxxvi].Chemical modification investigation of important residues begun [xxxvii,xxxviii]. Hepatitis Delta Virus (HDV) Ribozyme Size: ~60 nucleotides.Trans cleavage of target RNAs demonstrated [xxxix]. Binding sites andstructural requirements not fully determined, although no sequences 5′of cleavage site are required. Folded ribozyme contains a pseudoknotstructure [xl]. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. Only 2 known members of this class. Found in human HDV. Circularform of HDV is active and shows increased nuclease stability [xli]

REFERENCES

-   i. Michel, Francois; Westhof, Eric. Slippery substrates. Nat.    Struct. Biol. (1994), 1(1), 5-7.-   ii. Lisacek, Frederique; Diaz, Yolande; Michel, Francois. Automatic    identification of group I intron cores in genomic DNA sequences. J.    Mol. Biol. (1994), 235(4), 1206-17.-   iii. Herschlag, Daniel; Cech, Thomas R. Catalysis of RNA cleavage by    the Tetrahymena thermophila ribozyme. 1. Kinetic description of the    reaction of an RNA substrate complementary to the active site.    Biochemistry (1990), 29(44), 10159-71.-   iv. Herschlag, Daniel; Cech, Thomas R. Catalysis of RNA cleavage by    the Tetrahymena thermophila ribozyme. 2. Kinetic description of the    reaction of an RNA substrate that forms a mismatch at the active    site. Biochemistry (1990), 29(44), 10172-80.-   v. Knitt, Deborah S.; Herschlag, Daniel. pH Dependencies of the    Tetrahymena Ribozyme Reveal an Unconventional Origin of an Apparent    pKa. Biochemistry (1996), 35(5), 1560-70.-   vi. Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H. A    mechanistic framework for the second step of splicing catalyzed by    the Tetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58.-   vii. Li, Yi; Bevilacqua, Philip C.; Mathews, David; Turner,    Douglas H. Thermodynamic and activation parameters for binding of a    pyrene-labeled substrate by the Tetrahymena ribozyme: docking is not    diffusion-controlled and is driven by a favorable entropy change.    Biochemistry (1995), 34(44), 14394-9.-   viii. Banerjee, Aloke Raj; Turner, Douglas H. The time dependence of    chemical modification reveals slow steps in the folding of a group I    ribozyme. Biochemistry (1995), 34(19), 6504-12.-   ix. Zarrinkar, Patrick P.; Williamson, James R. The P9.1-P9.2    peripheral extension helps guide folding of the Tetrahymena    ribozyme. Nucleic Acids Res. (1996), 24(5), 854-8.-   x. Strobel, Scott A.; Cech, Thomas R. Minor groove recognition of    the conserved G.cntdot.U pair at the Tetrahymena ribozyme reaction    site. Science (Washington, D.C.) (1995), 267(5198), 675-9.-   xi. Strobel, Scott A.; Cech, Thomas R. Exocyclic Amine of the    Conserved G.cntdot.U Pair at the Cleavage Site of the Tetrahymena    Ribozyme Contributes to 5′-Splice Site Selection and Transition    State Stabilization. Biochemistry (1996), 35(4), 1201-11.-   xii. Sullenger, Bruce A.; Cech, Thomas R. Ribozyme-mediated repair    of defective mRNA by targeted trans-splicing. Nature (London)    (1994), 371(6498), 619-22.-   xiii. Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol. Chem.,    247, 5243-5251 (1972).-   xiv. Forster, Anthony C.; Altman, Sidney. External guide sequences    for an RNA enzyme. Science (Washington, D.C., 1883-) (1990),    249(4970), 783-6.-   xv. Yuan, Y.; Hwang, E. S.; Altman, S. Targeted cleavage of mRNA by    human RNase P. Proc. Natl. Acad. Sci. USA (1992) 89, 8006-10.-   xvi. Harris, Michael E.; Pace, Norman R. Identification of    phosphates involved in catalysis by the ribozyme RNase P RNA. RNA    (1995), 1(2), 210-18.-   xvii. Pan, Tao; Loria, Andrew; Zhong, Kun. Probing of tertiary    interactions in RNA: 2′-hydroxyl-base contacts between the RNase P    RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A. (1995), 92(26),    12510-14.-   xviii. Pyle, Anna Marie; Green, Justin B. Building a Kinetic    Framework for Group II Intron Ribozyme Activity: Quantitation of    Interdomain Binding and Reaction Rate. Biochemistry (1994), 33(9),    2716-25.-   xix. Michels, William J. Jr.; Pyle, Anna Marie. Conversion of a    Group II Intron into a New Multiple-Turnover Ribozyme that    Selectively Cleaves Oligonucleotides: Elucidation of Reaction    Mechanism and Structure/Function Relationships. Biochemistry (1995),    34(9), 2965-77.-   xx. Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang, Jian;    Perlman, Philip S.; Lambowitz, Alan M. A group II intron RNA is a    catalytic component of a DNA endonuclease involved in intron    mobility. Cell (Cambridge, Mass.) (1995), 83(4), 529-38.-   xxi. Griffin, Edmund A., Jr.; Qin, Zhifeng; Michels, Williams J.,    Jr.; Pyle, Anna Marie. Group II intron ribozymes that cleave DNA and    RNA linkages with similar efficiency, and lack contacts with    substrate 2′-hydroxyl groups. Chem. Biol. (1995), 2(11), 761-70.-   xxii. Michel, Francois; Ferat, Jean Luc. Structure and activities of    group II introns. Annu. Rev. Biochem. (1995), 64, 435-61.-   xxiii. Abramovitz, Dana L.; Friedman, Richard A.; Pyle, Anna Marie.    Catalytic role of 2′-hydroxyl groups within a group II intron active    site. Science (Washington, D.C.) (1996), 271(5254), 1410-13.-   xxiv. Daniels, Danette L.; Michels, William J., Jr.; Pyle, Anna    Marie. Two competing pathways for self-splicing by group II introns:    a quantitative analysis of in vitro reaction rates and products. J.    Mol. Biol. (1996), 256(1), 31-49.-   xxv. Guo, Hans C. T.; Collins, Richard A. Efficient trans-cleavage    of a stem-loop RNA substrate by a ribozyme derived from Neurospora    VS RNA. EMBO J. (1995), 14(2), 368-76.-   xxvi. Scott, W. G., Finch, J. T., Aaron, K. The crystal structure of    an all RNA hammerhead ribozyme: A proposed mechanism for RNA    catalytic cleavage. Cell, (1995), 81, 991-1002.-   xxvii. McKay, Structure and function of the hammerhead ribozyme: an    unfinished story. RNA, (1996), 2, 395-403.-   xxviii. Long, D., Uhlenbeck, O., Hertel, K. Ligation with hammerhead    ribozymes. U.S. Pat. No. 5,633,133.-   xxix. Hertel, K. J., Herschlag, D., Uhlenbeck, O. A kinetic and    thermodynamic framework for the hammerhead ribozyme reaction.    Biochemistry, (1994) 33, 3374-3385. Beigelman, L., et al., Chemical    modifications of hammerhead ribozymes. J. Biol. Chem., (1995) 270,    25702-25708.-   xxx. Beigelman, L., et al., Chemical modifications of hammerhead    ribozymes. J. Biol. Chem., (1995) 270, 25702-25708.-   xxxi. Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz,    Phillip. ‘Hairpin’ catalytic RNA model: evidence for helixes and    sequence requirement for substrate RNA. Nucleic Acids Res. (1990),    18(2), 299-304.-   xxxii. Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, John M.    Novel guanosine requirement for catalysis by the hairpin ribozyme.    Nature (London) (1991), 354(6351), 320-2.-   xxxiii. Berzal-Herranz, Alfredo; Joseph, Simpson; Chowrira, Bharat    M.; Butcher, Samuel E.; Burke, John M. Essential nucleotide    sequences and secondary structure elements of the hairpin ribozyme.    EMBO J. (1993), 12(6), 2567-73.-   xxxiv. Joseph, Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat    M.; Butcher, Samuel E. Substrate selection rules for the hairpin    ribozyme determined by in vitro selection, mutation, and analysis of    mismatched substrates. Genes Dev. (1993), 7(1), 130-8.-   xxxv. Berzal-Herranz, Alfredo; Joseph, Simpson; Burke, John M. In    vitro selection of active hairpin ribozymes by sequential    RNA-catalyzed cleavage and ligation reactions. Genes Dev. (1992),    6(1), 129-34.-   xxxvi. Hegg, Lisa A.; Fedor, Martha J. Kinetics and Thermodynamics    of Intermolecular Catalysis by Hairpin Ribozymes. Biochemistry    (1995), 34(48), 15813-28.-   xxxvii. Grasby, Jane A.; Mersmann, Karin; Singh, Mohinder; Gait,    Michael J. Purine Functional Groups in Essential Residues of the    Hairpin Ribozyme Required for Catalytic Cleavage of RNA.    Biochemistry (1995), 34(12), 4068-76.-   xxxviii. Schmidt, Sabine; Beigelman, Leonid; Karpeisky, Alexander;    Usman, Nassim; Sorensen, Ulrik S.; Gait, Michael J. Base and sugar    requirements for RNA cleavage of essential nucleoside residues in    internal loop B of the hairpin ribozyme: implications for secondary    structure. Nucleic Acids Res. (1996), 24(4), 573-81.-   xxxix. Perrotta, Anne T.; Been, Michael D. Cleavage of    oligoribonucleotides by a ribozyme derived from the hepatitis    .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.-   xl. Perrotta, Anne T.; Been, Michael D. A pseudoknot-like structure    required for efficient self-cleavage of hepatitis delta virus RNA.    Nature (London) (1991), 350(6317), 434-6.-   xli. Puttaraju, M.; Perrotta, Anne T.; Been, Michael D. A circular    trans-acting hepatitis delta virus ribozyme. Nucleic Acids Res.    (1993), 21(18), 4253-8.

TABLE II Reagent Equivalents Amount Wait Time* DNA Wait Time*2′-O-methyl Wait Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394Instrument Phosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-EthylTetrazole 23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL5 sec 5 sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 secBeaucage 12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NANA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 1531 μL 45 sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min465 sec Acetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124μL 5 sec 5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 secIodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300sec 300 sec Acetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle96 well Instrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Time* 2′-O-Reagent 2′-O-methyl/Ribo methyl/Ribo Wait Time* DNA methyl Wait Time*Ribo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl Tetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery.

TABLE III Peptides for Conjugation SEQ ID Peptide Sequence NO ANTENNAPRQI KIW FQN RRM KWK K amide 14 EDIA Kaposi AAV ALL PAV LLA LLA P + VQR15 fibroblast KRQ KLMP growth factor caiman MGL GLH LLV LAA ALQ GA 16crocodylus Ig(5) light chain HIVenvelope GAL FLG FLG AAG STM GA + PKS 17glycoprotein KRK 5 (NLS of the SV40) gp41 HIV-1 Tat RKK RRQ RRR 18Influenza GLFEAIAGFIENGWEGMIDGGGYC 19 hemagglutinin envelop glycoproteinRGD peptide X-RGD-X 20 where X is any amino acid or peptide transportanA GWT LNS AGY LLG KIN LKA LAA 21 LAK KIL Somatostatin (S)FC YWK TCT 22(tyr-3- octreotate) Pre-S-peptide (S)DH QLN PAF 23 (S) optional Serinefor coupling Italic = optional D isomer for stability

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5at 5′- S/AS end 1 at 3′- end “Stab 2” Ribo Ribo — All Usually ASlinkages “Stab 3” 2′-fluoro Ribo — 4 at 5′- Usually S end 4 at 3′- end“Stab 4” 2′-fluoro Ribo 5′ and — Usually S 3′-ends “Stab 5” 2′-fluoroRibo — 1 at 3′- Usually AS end “Stab 6” 2′-O-Methyl Ribo 5′ and —Usually S 3′-ends “Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually S 3′-ends“Stab 8” 2′-fluoro 2′-O- — 1 at 3′- Usually AS Methyl end “Stab 9” RiboRibo 5′ and — Usually S 3′-ends “Stab 10” Ribo Ribo — 1 at 3′- UsuallyAS end “Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′- Usually AS end “Stab 12”2′-fluoro LNA 5′ and Usually S 3′-ends “Stab 13” 2′-fluoro LNA 1 at 3′-Usually AS end “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′- Usually AS end 1 at3′- end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′- Usually AS end 1 at 3′- end“Stab 16” Ribo 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 17”2′-O-Methyl 2′-O- 5′ and Usually S Methyl 3′-ends “Stab 18” 2′-fluoro2′-O- 5′ and 1 at 3′- Usually S Methyl 3′-ends end “Stab 19” 2′-fluoro2′-O- 3′-end Usually AS Methyl “Stab 20” 2′-fluoro 2′-deoxy 3′-endUsually AS “Stab 21” 2′-fluoro Ribo 3′-end Usually AS “Stab 22” RiboRibo 3′-end- Usually AS CAP = any terminal cap, such as inverted deoxyabasic, glyceryl, or a conjugate moiety. All Stab 1-22 chemistries cancomprise 3′-terminal thymidine (TT) residues All Stab 1-22 chemistriestypically comprise 21 nucleotides, but can vary as described herein. S =sense strand AS = antisense strand

TABLE V Typical Hydrophobic Interaction Chromatography (HIC) Buffers pHConductivity Equilibrium 1.0 M  10 mM Sodium 7.0 ± 0.3 (A) AmmoniumPhosphate, Sulfate, Elution (B) N/A 100 mM Sodium 7.0 ± 0.3 Phosphate,Elution 2 (C) N/A N/A N/A <20 μS/cm

TABLE VI Typical HIC Gradient Gradient Step Buffer Time Step 1 100% A to100% B 20 cv Step 2 100% B to 20% C 30 cv Step 3  20% C to 100% C 30 cv

1. A compound having Formula 107:

wherein X comprises a short interfering nucleic acid (siNA) molecule;each W independently comprises a linker molecule or chemical linkageselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage, Y comprises a linkermolecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N, amide, alkylamine, aminoacid,polyamine, cyclic amine or heterocyclic amine, and Cholesterol comprisescholesterol.
 2. A compound having the formula below:

wherein X comprises a short interfering nucleic acid (siNA) molecule;each W independently comprises a linker molecule or chemical linkageselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage, Y comprises a linkermolecule that can be present or absent; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N, amide, alkylamine, aminoacid,polyamine, cyclic amine or heterocyclic amine; and n is independently aninteger from about 1 to about
 20. 3. A compound having Formula 111:

wherein X comprises a short interfering nucleic acid (siNA) moleculecapable of mediating RNA interference (RNAi); W comprises a linkermolecule or chemical linkage that can be present or absent, and whenpresent, forms only one covalent bond to the siNA molecule; and n is aninteger from about 1 to about
 20. 4. The compound of claim 1, wherein Wcomprises a linker molecule or chemical linkage selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.
 5. The compound of claim 3, wherein Wcomprises a linker molecule or chemical linkage selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.
 6. The compound of claim 1, wherein saidsiNA molecule comprises a sense strand and an antisense strand, andwherein said sense strand is conjugated with a compound comprisingFormula
 107. 7. The compound of claim 3, wherein said siNA moleculecomprises a sense strand and an antisense strand, and wherein said sensestrand is conjugated with a compound comprising Formula
 107. 8. Thecompound of claim 2, wherein W comprises a linker molecule or chemicallinkage selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.
 9. Thecompound of claim 2, wherein the siNA molecule comprises a sense strandand an antisense strand, and wherein the sense strand is conjugated witha compound of claim 2.